Activation and expansion of T-cells using an engineered multivalent signaling platform

ABSTRACT

The present invention relates generally to methods for activating and expanding cells, and more particularly, to a novel method to activate and/or stimulate cells using an engineered multivalent signaling platform. Compositions of cells activated and expanded by the methods herein are further provided.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates generally to methods forstimulating and activating cells, and more particularly, to methods toactivate and expand cells using an engineered multivalent signalingplatform. The present invention also relates to methods for generatingengineered multivalent signaling platform and methods of using same.

[0003] 2. Description of the Related Art

[0004] Immunotherapy involving the priming and expansion of Tlymphocytes (T cells) holds promise for the treatment of cancer andinfectious diseases, particularly in humans (Melief et al., Immunol.Rev. 145:167-177 (1995); Riddell et al., Annu. Rev. Immunol. 13:545-586(1995)). Current studies of adoptive transfer in patients with HIV, CMV,and melanoma involve the infusion of T cells that have been stimulated,cloned and expanded for many weeks in vitro on autologous dendriticcells (DC), virally infected B cells, and/or allogeneic feeder cells(Riddell et al., Science 257:238-241 (1992); Yee et al., J. Exp. Med.192:1637-1644 (2000); Brodie et al., Nat. Med. 5:34-41 (1999); Riddellet al., Hum. Gene Ther. 3:319-338 (1992), Riddell et al., J. Immunol.Methods 128:189-201 (1990)). However, adoptive T cell immunotherapyclinical trials commonly use billions of cells (Riddell et al., 1995).In order to produce these quantities of cells, many fold expansion of Tcells in vitro (40 population doublings) is usually required.Furthermore, for optimal engraftment potential and possible therapeuticbenefit, it is important to ensure that the T cells, after in vitroexpansion, are functional, and not senescent, at the time ofre-infusion.

[0005] Naturally occurring T cell activation is initiated by theengagement of the T cell receptor/CD3 complex (TCR/CD3) by apeptide-antigen bound to a major histocompatibility complex (MHC)molecule on the surface of an antigen-presenting cell (APC) (Schwartz,Science 248:1349 (1990)). While this is the primary signal in T cellactivation, other receptor-ligand interactions between APCs and T cellsare required for complete activation. For example, TCR stimulation inthe absence of other molecular interactions can induce a state ofanergy, such that these cells cannot respond to full activation signalsupon restimulation (Schwartz, 1990; Harding, et al., Nature 356:607(1992)). In the alternative, T cells die by programmed cell death(apoptosis) when activated by TCR engagement alone (Webb et al., Cell63:1249 (1990); Kawabe et al., Nature 349:245 (1991); Kabelitz et al.,Int. Immunol. 4:1381 (1992); Groux et al., Eur. J. Immunol. 23:1623(1993)).

[0006] Multiple receptor-ligand interactions take place between the Tcell and the APC, many of which are adhesive in nature, reinforcing thecontact between the two cells (Springer et al., Ann. Rev. Immunol. 5:223(1987)), while other interactions transduce additional activationsignals to the T cell (Bierer et al., Adv. Cancer Res. 56:49 (1991)).For example, CD28 is a surface glycoprotein present on 80% of peripheralT cells in humans and is present on both resting and activated T cells.CD28 binds to B7-1 (CD80) or B7-2 (CD86) and is the most potent of theknown co-stimulatory molecules (June et al., Immunol. Today 15:321(1994); Linsley et al., Ann. Rev. Immunol. 11:191 (1993)). CD28 ligationon T cells in conjunction with TCR engagement induces the production ofIL-2 molecules (June et al., 1994; Jenkins et al., 1993; Schwartz,1992). While the exact in vivo role of IL-2 is still in question, thereis little doubt that IL-2 is a critical factor for ex vivo T cellexpansion (Smith et al., Ann. N.Y. Acad. Sci. 332:423-432 (1979); Gilliset al., Nature 268:154-156 (1977)).

[0007] Recently, several new co-stimulatory molecules have beendiscovered based on their homology with the B7 and CD28 families. PD-1is expressed on activated T cells and has two B7 like ligands, PD-L1 andPD-L2. Presently, it is unclear whether PD-1 ligation delivers aninhibitory (Freeman et al., J. Exp. Med. 192:1027-1034 (2000); Latchmanet al., Nat. Immunol. 2:261-268 (2001)) or co-stimulatory signal (Donget al., Nat. Med. 5:1365-1369 (1999); Tseng et al., J. Exp. Med.193:839-846 (2001)) to T cells. B7-H3, which does not bind to CD28,CTLA-4, ICOS or PD-1, also reportedly acts as a co-stimulatory moleculefor T cell activation and IFN-γ production (Chapoval et al., Nat.Immunol. 2:269-274 (2001)).

[0008] The TNF receptor family member 4-1BB (CD137) was initiallyidentified in receptor screens of activated lymphocytes (Pollok,K. E. etal. Inducible T cell antigen 4-1BB. Analysis of expression and function.J. Immunol. 150, 771-781 (1993)). The 4-1BB ligand is expressed byactivated B cells, dendritic cells, and monocytes/macrophages, all ofwhich can act as APCs (Goodwin,R. G. et al. Molecular cloning of aligand for the inducible T cell gene 4-1BB: a member of an emergingfamily of cytokines with homology to tumor necrosis factor. Eur J.Immunol. 23, 2631-2641 (1993)). Previous studies have shown that 4-1BBis a co-stimulatory molecule in the activation of T cells, and itssignaling is independent from, albeit weaker than, CD28 signaling(Hurtado,J. C., Kim,Y J. & Kwon,B. S. Signals through 4-1BB arecostimulatory to previously activated splenic T cells and inhibitactivation-induced cell death. J. Immunol. 158, 2600-2609 (1997);Hurtado,J. C., Kim,S. H., Pollok,K. E., Lee,Z. H. & Kwon,B. S. Potentialrole of 4-1BB in T cell activation. Comparison with the costimulatorymolecule CD28. J. Immunol. 155, 3360-3367 (1995); Saoulli,K. et al.CD28-independent, TRAF2-dependent costimulation of resting T cells by4-1BB ligand. J. Exp. Med. 187, 1849-1862 (1998)). 4-1BB stimulationpreferentially activates CD8⁺ T cells in vitro and amplifies generationof CTL responses in vivo (Shuford,W. W. et al. 4-1BB costimulatorysignals preferentially induce CD8+ T cell proliferation and lead to theamplification in vivo of cytotoxic T cell responses. J. Exp. Med. 186,47-55 (1997)). The mechanism for this effect may involve improvedsurvival of activated CTLs (Takahashi,C., Mittler,R. S. & Vella,A. T.Cutting edge: 4-1BB is a bona fide CD8 T cell survival signal. J.Immunol. 162, 5037-5040 (1999)). Consistent with these data,co-stimulation of 4-1BB has been shown to have anti-viral and anti-tumoreffects (Tan,J. T. et al 4-1BB costimulation is required for protectiveanti-viral immunity after peptide vaccination. J. Immunol. 164,2320-2325 (2000); Melero,I. et al. Monoclonal antibodies against the4-1BB T cell activation molecule eradicate established tumors. Nat. Med.3, 682-685 (1997); Melero,I. et al. Amplification of tumor immunity bygene transfer of the co-stimulatory 4-1BB ligand: synergy with the CD28co-stimulatory pathway. Eur. J. Immunol. 28, 1116-1121 (1998);DeBenedette,M. A., Shahinian,A., Mak,T. W. & Watts,T. H. Costimulationof J. Immunol. 158, 551-559 (1997); Guinn,B. A., DeBenedette,M. A.,Watts,T. H. & Bernstein,N. L. 4-1BBL cooperates with B7-1 and B7-2 inconverting a B cell lymphoma cell line into a long-lasting antitumorvaccine. J. Immunol. 162, 5003-5010 (1999)).

[0009] Co-stimulation of T cells has been shown to affect multipleaspects of T cell activation (June et al., 1994). It lowers theconcentration of anti-CD3 required to induce a proliferative response inculture (Gimmi et al., Proc. Natl. Acad. Sci. USA 88:6575 (1991)). CD28co-stimulation also markedly enhances the production of lymphokines byhelper T cells through transcriptional and post-transcriptionalregulation of gene expression Lindsten et al., Science 244:339 (1989);Fraser et al., Science 251:313 (1991)), and can activate the cytolyticpotential of cytotoxic T cells. Inhibition of CD28 co-stimulation in tovivo can block xenograft rejection, and allograft rejection issignificantly delayed (Lenschow et al., Science 257:789 (1992); Turka etal., Proc. Natl. Acad. Sci. USA 89:11102 (1992)).

[0010] Methods of expanding T cell clones and/or lines for adoptiveimmunotherapy have proven to have certain drawbacks. The standardculture of pure CD8⁺ cells is limited by apoptosis, diminution ofbiological function and/or proliferation, and obtaining a sufficientnumber of cells to be useful has been particularly difficult. Currentcell culture techniques require several months to produce sufficientnumbers of cells from a single clone (Riddell et al., 1992; Heslop etal., Nat. Med. 2:551-555 (1996)), which is a problematic limiting factorin the setting of malignancy. Indeed, it is possible that such the Tcells that are currently infused into patients, may have a limitedreplicative capacity, and therefore, could not stably engraft to providelong-term protection from disease. Furthermore, the various techniquesavailable for expanding human T cells have relied primarily on the useof accessory cells (i.e. cells that support or promote T cell survivaland proliferation such as PBMC or DC, B cells, monocytes, etc.) and/orexogenous growth factors, such as interleukin-2 (IL-2). IL-2 has beenused together with an anti-CD3 antibody to stimulate T cellproliferation. Both primary and secondary APC signals are thought to berequired for optimal T cell activation, expansion, and long-termsurvival of the T cells upon re-infusion. The requirement for accessorycells presents a significant problem for long-term culture systemsbecause these cells are relatively short-lived. Therefore, in along-term culture system, APCs must be continually obtained from asource and replenished. The necessity for a renewable supply ofaccessory cells is problematic for treatment of immunodeficiencies inwhich accessory cells are affected. In addition, when treating viralinfection, if accessory cells carry the virus, the cells may contaminatethe entire T cell population during long-term culture.

[0011] In the absence of exogenous growth factors or accessory cells, aco-stimulatory signal may be delivered to a T cell population, forexample, by exposing the cells to a CD3 ligand and a CD28 ligandattached to a solid phase surface, such as a bead. See C. June, et al.(U.S. Pat. Nos. 5,858,358 and 6,352,694); C. June et al. WO 99/953823.While these methods are capable of achieving therapeutically useful Tcell populations, increased robustness and ease of T cell preparationremain less than ideal.

[0012] In addition, the methods currently available in the art have notfocused on obtaining a more robust population of T cells and thebeneficial results thereof. Furthermore, the applicability of activatedand expanded T cells has been limited to only a few disease states. Formaximum in vivo effectiveness, theoretically, an ex vivo- or invivo-generated, activated T cell population should be in a state thatcan maximally orchestrate an immune response to cancer, infectiousdisease, or other disease states. While previous investigators havenoted long term qualitative persistence of T cells in human adoptivetransfer protocols, the quantitative level of sustained engraftment hasbeen low (Rosenberg et al., N. Engl. J. Med. 323:570-578 (1990); Dudleyet al., J. Immunother. 24:363-373 (2001); Yee et al., Curr. Opin.Immunol. 13:141-146 (2001); Rooney et al., Blood 92:1549-1555 (1998)).

[0013] Therefore, the present invention offers therapeutic advantagesbecause there remains an unmet need for sustained high-level engraftmentof human T lymphocytes. Methods of stimulating the expansion of certainsubsets of T cells have the potential to generate a variety of T cellcompositions useful in immunotherapy. Successful immunotherapy can beaided by increasing the reactivity and quantity of T cells by efficientstimulation. The present invention provides methods to generate anincreased number of activated and pure T cells that have surfacereceptor and cytokine production characteristics that are optimal for Tcell-mediated immune responses and that appear more physiologicallyfunctional than T cells produced by other expansion methods. Inaddition, the present invention provides compositions of cellpopulations of any target cell, including T cell populations andparameters for producing the same, as well as providing other relatedadvantages.

BRIEF SUMMARY OF THE INVENTION

[0014] The present invention provides engineered multivalent signalingplatforms for use in stimulation and/or expanding T-cells. Also providedare methods of using the EMSPs either to stimulate or expand T-cells exvivo or in vivo or a combination thereof, such as stimulating ex vivoand allowing for expansion in vivo. To this end, one of ordinary skillin the art would understand that a variety of combinations of theelements of the present invention is easily identifiable. For instance,T-cells either stimulated and/or expanded can be used for therapeuticpurposes. Moreover, it would be clear upon reference to the instantspecification that EMSPs may also be administered to a patient in vivothereby stimulating and/or expanding T-cells in vivo. Such in vivo usesare numerous, including breaking tolerance to an antigen from a tumor orpathogen or as a means to enhance antigen reactivity of a vaccinethereby acting as an adjuvant.

[0015] In one aspect, the present invention is directed to stimulation,activation, or expansion of T-cells, including but not limited to CD4+and CD8+ T-cells. Further, the present invention finds particularbenefit in the ability to sequentially stimulate and expand CD8⁺ cellswithout a significant loss in viability and maintaining function of theCD8+ T-cells after multiple rounds of stimulation with EMSPs.

[0016] In another aspect, the present invention provides an engineeredmultivalent signaling platform (EMSP) for use in stimulating and/oractivating T cells comprising an EMSP that expresses or displays on itssurface one or more agents that ligate a cell surface moiety of at leasta portion of T-cells and stimulates said T-cells. In certain embodimentsthis platform may comprise a cell line. This cell line may by mammalianincluding human. In certain embodiments the cell line displays low or noendogenous MHC as compared with typical antigen presenting cells. Inrelated embodiments, the human cell line may be K562, U937, 721.221, T2,and C1R cells.

[0017] In related aspects, the EMSPs of the present invention are cellsthat are genetically modified to express a human Fcγ receptor ormanipulated to have this receptor bound to the surface of the EMSP. Incertain embodiments the receptor comprises CD32. In other embodiments,the EMSP is a cell that is genetically modified to express CD32 and saidone or more agent is an antibody that binds to a cell surface moleculeon the surface of T cells. Also provided are the presently describedEMSPs wherein the EMSP is further genetically modified to express ormanipulated to display a co-stimulatory molecule for a T-cell. Incertain embodiments the co-stimulatory molecule may be any one of or acombination of CD80, CD86, 4-1BBL, OX40L, ICOS-L, ICAM, PD-L1 and PD-L2.Other embodiments include but are not limited to EMSPs wherein one ormore agent on the surface is an antibody that is displayed on thesurface of said EMSP via interaction with the Fcγ receptor. Otherembodiments, of course include wherein the one or more agent displayedor expressed on the EMSP is a natural ligand for a T-cell such as thoseto CD28 and 4-1BB.

[0018] In certain aspects the present invention also provides EMSPs inthe form of cells or cell lines that have been genetically modified toexpress stimulatory agents, co-stimulatory agents, and/or cytokines aswell as other polypeptides. When cytokines are expressed any of thosedesired by be utilized. For example, IL-2, IL-15, GM-CSF, IL-4, TNF-α,and/or IFN-γ be utilized among others.

[0019] In yet additional embodiments, the source of T-cells to bestimulated, activated, and/or expanded by use of the EMSPs may be anytype of T-cell, including CD4+, CD8+, regulatory T-cells and the like.

[0020] Also provided by the instant invention are EMSPs that display orexpress on their surface antibodies such as anti-CD3 and anti-CD28antibodies as well as other ligands and stimulatory or co-stimulatorymolecules such as 4-1BB ligand.

[0021] In other aspects the present invention provides methods foractivating or stimulating a population of T-cells by cell surface moietyligation, comprising providing a population of cells wherein at least aportion thereof comprises T-cells; contacting said population of cellswith an EMSP, said EMSP having on its surface one or more agents thatligate a cell surface moiety of at least a portion of said T-cells andactivates or stimulates said T-cells. In certain embodiments the EMSPcomprises a cell. In related embodiments the cell may be mammalian, thecell may be human, the cell may be murine, the cell may be from a cellline such as a human cell line. In further embodiments the T-cells areexpanded by culturing the cells following stimulation with EMSPs underconditions and for time sufficient to provide expansion. Such expansionmay occur in the presence or absence of EMSPs as well as in the presenceor absence of exogenously added cytokines. Further, such stimulationand/or expansion may occur in vivo. In related embodiments the methodincludes separating said T-cells from said EMSP and subsequentlyincubating said T-cells with an agent that facilitates T-cell expansion.Those of skill in the art would appreciate that the T-cells stimulatedor expanded may be derived from any source such as from a patient, froma T-cell line, from a T-cell clone.

[0022] In yet additional aspects the present invention provides methodsfor maintaining T-cell repertoire, such as the Vbeta repertoire ofselect T-cell population to be expanded by stimulating with the EMSPs ofthe invention for a time sufficient to induce activation andsubsequently expanding said T-cells. In certain embodiments theexpansion occurs in the presence or absence of EMSPs.

[0023] In further related aspects, the EMSPs of the present inventionmay be used to maintain the viability of T-cells during culture, evenfollowing multiple/sequential rounds of stimulation. In certainembodiments sequential rounds of stimulation may be initiated by EMSPs.In certain aspects, even following sequential stimulation of T-cells,such as without limitation CD8+ T-cells, the viability of said T-cellsis greater than 50%, 60%, 70%, 80%, or 90% following at least one, two,three, four, five, six, seven, or eight rounds of stimulation.

[0024] Also provided are methods for decreasing apoptosis of apopulation of CD8+ T-cells, comprising stimulating said cells with EMSP,said EMSP having on its surface at least one primary stimulatory agentand at least one co-stimulatory agent. In certain embodiments, the CD8+T-cells are expanded in the presence or absence of said EMSP. In furtherembodiments, the CD8+ T-cells demonstrate an increase in Bcl-X_(L)levels compared to CD8+ T-cells expanded in the absence of initialstimulation with said EMSP. In yet additional embodiments the EMSPexpresses or displays a 4-1BB ligand. In other embodiments the EMSPdisplays or expresses an agent that binds to CD3, and agent that bindsto CD28 or an agent that binds 4-1BB or any combination or multiplethereof. In related embodiments sequential stimulation with the EMSPalso demonstrates decreased apoptosis as compared to cells sequentiallystimulated by other means such as by anti-CD3 and anti-CD28 coatedbeads.

[0025] In further aspects the present invention also provides methodsfor expanding a population of T-cells by cell surface moiety ligation,comprising providing a population of cells wherein at least a portionthereof comprises T-cells either in vivo or ex vivo; contacting saidpopulation of cells with an EMSP, said EMSP having on its surface one ormore agents that ligate a cell surface moiety of at least a portion ofsaid T-cells and activates or stimulates said T-cells; and culturingsaid T-cells under conditions and time sufficient to induce celldivision, wherein said contacting and/or said culturing occurs in theabsence of exogenously added cytokines. In yet additional embodimentsthe EMSP comprises a cell, such as a mammalian cell (e.g., human, mouse,etc.). The cell may be from a cell line. In some embodiments, at leastone round of sequential stimulation is provided. In related embodimentssequential rounds of stimulating said T-cells are performed with EMSPeither by previously purifying T-cells from originally added EMSP andsubsequently adding additional EMSP or by adding additional EMSP topreviously stimulated cells without separation of originally added EMSP.In other embodiments, the T-cells are separated from said EMSP andsubsequently incubated with an agent that facilitates T-cell expansion,followed by restimulation with EMSP. Of course the T-cells for thesemethods could be derived from any source including PBMC, purifiedT-cells, T-cell lines, T-cell clones, etc. In certain embodiments theEMSP comprises a cell displaying ligands for any one of CD3, CD28, or4-1BB including any combination thereof, such as ligands to all three.

[0026] In other aspects, the present invention provides methods forexpanding a population of T-cells by cell surface moiety ligation,comprising providing a population of cells wherein at least a portionthereof comprises T-cells either ex vivo or in vivo; contacting saidpopulation of cells with an EMSP, said EMSP having on its surface one ormore agents that ligate a cell surface moiety of at least a portion ofsaid T-cells and activates or stimulates said T-cells; and culturingsaid T-cells under conditions and time sufficient to induce celldivision. As with all the described methods herein in relatedembodiments the EMSP may comprise a cell such as a mammalian (e.g.,human or mouse cell or the like), a cell line or cell fusion or cellhybrid. In additional embodiments, the method further comprisessequentially stimulating said T-cells with EMSP either by previouslypurifying T-cells from originally added EMSP and subsequently addingadditional EMSP or by adding additional EMSP to previously stimulatedcells without separation of originally added EMSP. In a relatedembodiment, the methods comprise separating said T-cells from said EMSPand subsequently incubating said T-cells with an agent that facilitatesT-cell expansion, followed by restimulation with EMSP. In certainembodiments the EMSP comprises a cell displaying ligands for any one ofCD3, CD28, or 4-1BB including any combination thereof, such as ligandsto all three. In yet additional embodiments, following initialstimulation or activation said T-cells are separated from EMSP and thenexpanded in the presence of exogenously added cytokines. In yet still anadditional embodiment the methods of the present invention include theprevious method may include T-cells that are initially stimulated byEMSPs but that are substantially free of EMSP prior to expansion.

[0027] One aspect of the present invention provides a population ofT-cells expanded by the methods of the present invention either in theabsence or presence of exogenous cytokines and wherein said T-cells aresubstantially free of EMSP.

[0028] An additional aspect of the present invention provide for amethod of immunotherapy comprising administering an EMSP to a subject inneed thereof, thereby stimulating a population of T-cells within thesubject to expand.

[0029] A further aspect of the present invention comprises a method ofenhancing reactivity to an antigen comprising administering an EMSP to asubject in need thereof concurrently, prior to or following inoculationof the subject with an antigen of interest, thereby stimulating apopulation of antigen specific T-cells within the subject to expand.

[0030] Yet a further aspect of the present invention comprises a methodfor breaking immune tolerance comprising administering an EMSP to asubject either systemically or locally at a site of interest.

[0031] One additional aspect of the invention comprises a method forincreasing uptake of an exogenously added nucleic acid molecule inT-cells, comprising contacting said T-cells with an EMSP and contactingsaid T-cells with said nucleic acid molecule thereby, said contacting ofEMSP with said T-cells rendering cells more amenable to uptake ofnucleic acid. In one embodiment of the invention, exogenously addednucleic acids are operably linked to a promoter. In certain embodiments,nucleic acid molecules provided herein provide gene replacement forabnormal gene products.

[0032] In certain aspects of the present invention, the naturalfunctionality of said T-cells is preserved following stimulation andexpansion using the methods provided herein.

[0033] In another aspect fo the invention, a method of activatingantigen specific T-cells is provided comprising contacting a populationof T-cells an antigen and an EMSP under conditions and for timesufficient to induce activation of T-cells specific to said antigen. Incertain embodiments, activation occurs ex vivo after which the T-cellsare administered to a patient substantially free or in combination withsaid EMSP. In one embodiment, the antigen is displayed by said EMSP. Ina particular embodiment, the antigen is displayed by way of a fusionprotein. In another embodiment, the antigen is the form of a complexwith an MHC molecule or MHC derived peptide such as in an peptide-MHCtetramer, dimer, or monomer.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0034] The foregoing summary, as well as the following detaileddescription of the invention, will be better understood when read inconjunction with the appended drawings. For the purpose of illustratingthe invention, there are shown in the drawings, certain embodiment(s)that are presently preferred. It should be understood, however, that theinvention is not limited to the precise arrangements andinstrumentalities shown.

[0035] FIGS. 1A-1C depict construction of artificial APCs (aAPC) (anillustrative example of an engineered multivalent signaling platform, orEMSP) from the parental K562 cell line. FIG. 1A depicts two-colorflow-cytometric analysis of MHC I and II expression and CD54 and CD58expression in parental K562 cells (top panels). Expression of CD32 and4-1BBL in K32 (left) and K32/4-1BBL (right) cell lines is shown (middlepanels). Isotype controls for the anti-CD32 antibody (IgG2a) andanti-41BBL antibody (IgG1) are shown for each aAPC (bottom panels). FIG.1B depicts the engineered K32/4-1BBL aAPC interacting with a CD8+ Tcell. FIG. 1C graphically depicts proliferation of polyclonal CD4⁺ andCD8⁺ T cells stimulated with the indicated aAPCs, measured by[3H]thymidine incorporation between days 3 and 4 culture. T cells werestimulated with aAPCs as indicated, in the absence of cytokines. At 72hours the cells were pulsed with [3H]thymidine and incubated for anadditional 18 hours before harvesting. Counts per minute values areshown as mean±s.e.m. from triplicate cultures.

[0036] FIGS. 2A-2C depict long-term growth of primary polyclonal humanCD8⁺ T cells stimulated with aAPCs in the absence of exogenouscytokines. FIG. 2A graphically depicts CD8⁺ T cells stimulated withCD3/28 beads (X), irradiated K32 cells loaded with CD3/28 antibodies(A), or with irradiated K32/4-1BBL cells loaded with CD3/28 antibodies(). T cells were stimulated with aAPCs on days 0, 10, and 20 ofculture. FIGS. 2B, 2C depict the purity of T cells and the fate ofirradiated K32/4-1BBL stimulator cells assessed by staining for CD3, CD8(FIG. 2B), and CD32 (FIG. 2C) expression during the first 7 days ofculture. Variable numbers of red blood cells and platelets werecontained in the input cultures; gating on cell size/debris was not usedin this experiment so that all cells in the culture were represented.Viable cells are indicated by gating on propidium iodide to exclude deadcells. Results are representative of >10 different experiments, eachwith a different donor.

[0037] FIGS. 3A-3D depict propagation of antigen-specific cytotoxic Tcells from an HLA A*0201 donor using K32/4-1BBL aAPCs. FIG. 3A is aschematic of the experimental protocol of the present invention. FIG. 3Bdepicts the specificity of cell cultures as assessed by MHC tetramerstaining. CD8+ T cells were stained with anti-CD8 antibody (x-axis) andA*0201 tetrameric MHC (y-axis) loaded with influenza matrix proteinpeptide (fluMP). Left panel of 3B: initial cell population of T cells onday 0, with gates showing the cells into CD8⁺flu-tet⁺ and CD8⁺flu-tet⁻populations. Right panels of 3A: tetramer staining of CD8⁺flu-tet⁺ (top)or CD8⁺flu-tet⁻ (bottom) cultures after expansion on K32/4-1BBL cellsfor 26 days. FIG. 3C graphically depicts a growth curve of the sortedCD8⁺ T cell populations. T cells were sorted into CD8⁺ fluMP tetramer⁺() or CD8⁺ fluMP tetramer⁻ ( ). The sorted T cell populations were thenstimulated with irradiated K32/4-1BBL cells loaded with CD3/28antibodies as indicated (arrows). rIL-2 was added to the culturesbeginning on day 28. The total cell numbers are depicted in a semi-logplot of cell number v. days in culture. FIG. 3D graphically depictscytotoxicity of flu-specific T cells after expansion on K32/4-1BBL aAPCsfor 26 days. ⁵¹Cr-release assays were done using TAP-deficient HLAA*0201 T2 target cells pulsed (circles) or unpulsed (squares) with thefluMP peptide. Antigen-specific cytotoxicity was also examined bycomparing CD8⁺ fluMP tetramer⁺ cells (closed symbols) to CD8⁺FluMPtetramer cells (open symbols). Values shown as mean±s.e.m. of triplicatecultures. Y-axis, percentage of specific 51Cr release; x-axis,effector:target (E:T) ratios. The entire protocol is representative ofthree experiments, each from different donors.

[0038]FIG. 4 depicts maintenance of the TCR Vβ repertoire in polyclonalCD8⁺ T cells after expansion with K32/4-1BBL aAPCs. T cells cultured onK32 or K32/4-1BBL aAPCs from the growth curve shown in FIG. 2 wereassessed for the CDR3 length distribution. The indicated TCR Vβ familyis shown at baseline, and after 17 days of culture.

[0039]FIGS. 5A and 5B graphically depict expression of genes involved inT cell growth and survival after stimulation with aAPCs. Real-timequantitative RT-PCR of Bcl-xL (mean±s.e.m.) (FIG. 5A) or IL-2 (FIG. 5B)mRNA in polyclonal CD8⁺ T cultures. Y-axis:—fold expression of Bcl-xL orIL-2 relative to day 0 of culture. All cultures were stimulated withaAPCs on days 0 and 10. Results are representative of three differentexperiments with different donors.

[0040]FIG. 6 depicts the distinct effects on apoptosis in cultures ofpolyclonal human CD8⁺ T cells stimulated with various aAPCs.Flow-cytometric analysis of cultured cells stained with FITC-labeledannexin V (x-axis) and propidium iodide (y-axis). The three rowsrepresent different aAPCs used for stimulation. The columns representdays in culture. All cultures were stimulated with aAPCs on days 0 and10. Data shown are not gated. Results are representative of threeexperiments with different donors.

DETAILED DESCRIPTION OF THE INVENTION

[0041] Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms thatwill be used hereinafter.

[0042] The term “biocompatible”, as used herein, refers to the propertyof being predominantly non-toxic to living cells.

[0043] The term “stimulation”, as used herein, refers to a primaryresponse induced by ligation of a cell surface moiety. For example, inthe context of receptors, such stimulation entails the ligation of areceptor and a subsequent signal transduction event. With respect tostimulation of a T cell, such stimulation refers to the ligation of a Tcell surface moiety that in one embodiment subsequently induces a signaltransduction event, such as binding the TCR/CD3 complex. Further, thestimulation event may activate a cell and upregulate or downregulateexpression or secretion of a molecule, such as downregulation of TGF-β.Thus, ligation of cell surface moieties, even in the absence of a directsignal transduction event, may result in the reorganization ofcytoskeletal structures, or in the coalescing of cell surface moieties,each of which could serve to enhance, modify, or alter subsequentcellular responses.

[0044] The term “activation”, as used herein, refers to the state of acell following sufficient cell surface moiety ligation to induce anoticeable biochemical or morphological change. Within the context of Tcells, such activationrefers to the state of a T cell that has beensufficiently stimulated to induce cellular proliferation. Activation ofa T cell may also induce cytokine production and performance ofregulatory or cytolytic effector functions. Within the context of othercells, this term infers either up or down regulation of a particularphysico-chemical process The term “activated T cells” indicates T cellsthat are currently undergoing cell division, cytokine production,performance of reg or cytol. effector functions, and/or has recentlyundergone the process of “activation.”

[0045] The term “target cell”, as used herein, refers to any cell thatis intended to be stimulated by cell surface moiety ligation.

[0046] An “antibody”, as used herein, includes both polyclonal andmonoclonal antibodies; primatized (i.e., modified to include moreprimate-specific residues in a constant region) (e.g., humanized);murine; mouse-human; mouse-primate; and chimeric; and may be an intactmolecule, a fragment thereof (such as scFv, Fv, Fd, Fab, Fab′ andF(ab)′₂ fragments), or multimers or aggregates of intact moleculesand/or fragments; and may occur in nature or be produced, e.g., byimmunization, synthesis or genetic engineering; an “antibody fragment,”as used herein, refers to fragments, derived from or related to anantibody, which bind antigen and which in some embodiments may bederivatized to exhibit structural features that facilitate clearance anduptake, e.g., by the incorporation of galactose residues. This includes,e.g., F(ab), F(ab)′₂, scFv, light chain variable region (V_(L)), heavychain variable region (V_(H)), and combinations thereof.

[0047] The term “protein”, as used herein, includes proteins,polypeptides and peptides; and may be an intact molecule, a fragmentthereof, or multimers or aggregates of intact molecules and/orfragments; and may occur in nature or be produced, e.g., by synthesis(including chemical and/or enzymatic) or genetic engineering.

[0048] The term “T cell clone” as used herein includes T cells derivedfrom a single T cell or have identical TCRs. T cells can be cloned usingnumerous assays know in the art including limiting dilution assays (LDA)and cell sorting using flow cytometry.

[0049] The term “T cell line” as used herein includes T cell clones andmixed populations of T cells with different TCRs all of which mayrecognize the same target (e.g. antigen, tumor, virus).

[0050] The term “substantially free of” as used herein means apopulation of cells, e.g. T cells, that is at least 50% free of non-Tcells, or in certain embodiments at least 60, 70, 80, 85, or 90% free ofnon-T cells.

[0051] The term “agent”, “ligand”, or “agent that binds a cell surfacemoiety”, as used herein, refers to a molecule that binds to a definedpopulation of cells. The agent may bind any cell surface moiety, such asa receptor, an antigenic determinant, or other binding site present onthe target cell population. The agent may be a protein, peptide,antibody and antibody fragments thereof, fusion proteins, syntheticmolecule, an organic molecule (e.g., a small molecule), a carbohydrate,or the like. Within the specification and in the context of T cellstimulation, antibodies and natural ligands (e.g., B7 and 4-1BBL) areused as prototypical examples of such agents.

[0052] The terms “agent that binds a cell surface moiety” and “cellsurface moiety”, as used herein, are used in the context of aligand/anti-ligand pair. Accordingly, these molecules should be viewedas a complementary/anti-complementary set of molecules that demonstratespecific binding, generally of relatively high affinity (an affinityconstant, K_(a,) of about 10⁶ M⁻¹).

[0053] A “co-stimulatory signal”, as used herein, refers to a signal,which in combination with a primary signal, such as TCR/CD3 ligation,leads to T cell proliferation and/or upregulation or downregulation ofkey molecules.

[0054] A “ligand/anti-ligand pair”, as used herein, refers to acomplementary/anti-complementary set of molecules that demonstratespecific binding, generally of relatively high affinity (an affinityconstant, K_(a,) of about 10⁶ M⁻¹,). Exemplary ligand/anti-ligand pairsenzyme/inhibitor, hapten/antibody, lectin/carbohydrate, ligand/receptor,and biotin/avidin or streptavidin. Within the context of the presentinvention specification receptors and other cell surface moieties areanti-ligands, while agents (e.g., antibodies and antibody fragments)reactive therewith are considered ligands.

[0055] “Separation”, as used herein, includes any means of substantiallypurifying one component from another (e.g., by filtration or magneticattraction).

[0056] “Quiescent”, as used herein, refers to a cell state wherein thecell is not actively proliferating.

[0057] A “surface”, as used herein, refers to any surface capable ofhaving an agent attached thereto and includes, without limitation,metals, glass, plastics, co-polymers, colloids, lipids, lipid bilayers,cell surfaces such as EMSP as described herein, and the like.Essentially any surface that is capable of retaining an agent bound orattached thereto. A prototypical example of a surface used herein, is anengineered multivalent signaling platform or a particle such as a bead.

[0058] Generation of Engineered Multivalent Signaling Platforms (EMSP)

[0059] One aspect of the present invention is directed to the findingthat a cell-based universal engineered multivalent signaling platform(EMSP) specifically optimized for rapid expansion of human T cells canbe used to stimulate the long-term growth of functional polyclonal andantigen-specific human T lymphocytes. In particular, in one aspect ofthe present invention, the EMSP can be generated that stimulate thelong-term growth of CD8⁺ T cells. In another aspect of the presentinvention, the EMSP can be generated that stimulate the long-term growthof CD4+ T cells. In a further embodiment, the EMSP can be generated tostimulate the long-term growth of regulatory T cells. Similarly, theEMSP of the present invention can be used for stimulation of growth ofcombinations of T cell subsets (e.g. γδ-T cells, CD4+ and CD8+ ag-spec Tcells). In yet another embodiment, an EMSP can be generated to stimulateCD4/CD8 double positive T cells, or CD28-negative T cells. Oneillustrative embodiment of an EMSP is referred to as an aritificialantigen presenting cell (aAPC) and is described herein in the Examples.

[0060] An “engineered multivalent signaling platform (EMSP)”, as usedherein, refers to a lipid bilayer engineered (e.g., genetically,physically, or chemically manipulated) to have on its surface at leastone molecule capable of binding to a T-lymphocyte and inducing a primaryactivation event and/or a proliferative response or capable of binding amolecule having such an affect thereby acting as a scaffold. In oneembodiment, the EMSP is engineered to express a molecule that binds tothe Fc portion of an antibody. In an additional embodiment, an EMSPcomprises a cell line engineered to stably express a molecule capable ofbinding to the Fc portion of an antibody. This universal EMSP can thenbe loaded with any variety of antibodies that recognize cell surfacemolecules present on the surface of T lymphocytes, e.g. CD3, CD28,4-1BB, TCR, etc. In an alternative embodiment, an EMSP can be generatedby directly engineering a cell line to stably express the ligands forcell surface molecules present on the surface of T lymphocytes, e.g.CD3, CD28, 4-1BB, TCR, etc. The EMSP can be further engineered to stablyexpress one or more co-stimulatory molecules, for example CD86 or 4-1BBligand. For example, in one illustrative embodiment of the presentinvention, an EMSP is engineered to express the human low-affinity Fcγreceptor, CD32 and the CD86 molecule. In another illustrative embodimentof the present invention an EMSP is engineered to express CD32 and the4-1BB ligand. The skilled artisan would readily recognize that anyvariety and combination of co-stimulatory molecules can be used in thecontext of the present invention. Further, EMSP may be engineered toexpress a variety of molecules useful for the stimulation and activationof T lymphocytes and/or be loaded with a variety molecules useful forthe stimulation and activation of T lymphocytes. In some instances, theexpression of these ligands/receptors could be regulated by “regulatabletranscription promoters”, such a tetracycline dependent promoter, whichcould be an advantage in certain in vivo applications of the K32 line(see Examples) and its derivatives.

[0061] In a further embodiment, the EMSP of the present invention areengineered to express an antigen of interest, such as a tumor antigen(e.g. a melanoma, breast tumor, leukemia or other tumor antigen), anauto-antigen (e.g. MBP), a viral antigen (e.g. an HIV, CMV, EBV, orHepatitis antigen) or antigen of other pathogens of interest, presentedon the EMSP surface in the context of MHC. Alternatively, the EMSP ofthe present invention can be pulsed with antigen using any number ofassays known to the skilled artisan, , or transduced or otherwiseexpress MHC which can then be pulsed with peptide/antigen. In yetanother embodiment, an EMSP of the present invention can be engineeredto express peptide-MHC tetramers (Altman, et al., Science. 1996 October4;274(5284):94-6.), or monomers or dimers or trimers. Other illustrativemolecules and methods useful in the context of this invention are asdescribed in U.S. Pat. Nos. 6,001,365, 6,355,479, 5,529,921, and6,464,973, herein incorporated by reference in their entirety.

[0062] According to certain methods of the invention, antigen maycomprise defined tumor antigens such as, but not limited to, themelanoma antigen Melan-A (also referred to as melanoma antigenrecognized by T cells or MART-1), melanoma antigen-encoding genes 1, 2,and 3 (MAGE-1, -2, -3), melanoma GP100, carcinoembryonic antigen (CEA),the breast cancer angtigen, Her-2/Neu, serum prostate specific antigen(PSA), Wilm's Tumor (WT-1), mucin antigens, MUC-1, -2, -3, -4, and Bcell lymphoma idiotypes.

[0063] Antigen source may also comprise non-transformed, transformed,transfected, or transduced cells or cell lines. Cells may betransformed, transfected, or transduced using any of a variety ofexpression or retroviral vectors known to those of ordinary skill in theart that may be employed to express recombinant antigens. Expression mayalso be achieved in any appropriate host cell that has been transformed,transfected, or transduced with an expression or retroviral vectorcontaining a DNA molecule encoding recombinant antigen(s). Any number oftransfection, transformation, and transduction protocols known to thosein the art may be used, for example those outlined in Current Protocolsin Molecular Biology, John Wiley & Sons, New York, N.Y., or in numerouskits available commercially (e.g., Invitrogen Life Technologies,Carlsbad, Calif.). In one embodiment of the present invention,recombinant vaccinia vectors and cells infected with said vacciniavectors, may be used as a source of antigen. Recombinant antigen mayinclude any number of defined tumor antigens described below.

[0064] The EMSP of the present invention may be loaded with antigen orengineered to express a variety of stimulatory, co-stimulatorymolecules, targeting agents, and/or cytokines through geneticmodification. Genetic modification may comprise RNA or DNA transfectionusing any number of techniques known in the art, for exampleelectroporation (using e.g., the Gene Pulser II, BioRad, Richmond,Calif.), various cationic lipids, (LIPOFECTAMINE™, Life Technologies,Carlsbad, Calif.), or other techniques such as calcium phosphatetransfection as described in Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y. For example, 5-50 μg of RNA or DNA in500 μl of Opti-MEM can be mixed with a cationic lipid at a concentrationof 10 to 100 μg, and incubated at room temperature for 20 to 30 minutes.Other suitable lipids include LIPOFECTIN™, LIPOFECTAMINE™. The resultingnucleic acid-lipid complex is then added to 1-3×10⁶ cells, preferably2×10⁶, EMSP in a total volume of approximately 2 ml (e.g., in Opti-MEM),and incubated at 37° C. for 2 to 4 hours. The EMSP may also betransduced using viral transduction methodologies as described below.

[0065] The EMSP may alternatively be genetically engineered to express avariety of stimulatory molecules, co-stimulatory molecules, cytokines,and/or antigens using retroviral transduction technologies. In oneaspect of the invention, the retroviral vector may be an amphotropicretroviral vector, preferably a vector characterized in that it has along terminal repeat sequence (LTR), e.g., a retroviral vector derivedfrom the Moloney murine leukemia virus (MoMLV), myeloproliferativesarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murinestem cell virus (MSCV), spleen focus forming virus(SFFV), oradeno-associated virus (AAV). Most retroviral vectors are derived frommurine retroviruses. Retroviruses adaptable for use in accordance withthe present invention can, however, be derived from any avian ormammalian cell source. These retroviruses are preferably amphotropic,meaning that they are capable of infecting host cells of severalspecies, including humans. In one embodiment, the gene to be expressedreplaces the retroviral gag, pol and/or env sequences. A number ofillustrative retroviral systems have been described (e.g., U.S. Pat.Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman, BioTechniques7:980-90, 1989; Miller, A. D., Human Gene Therapy 1:5-14, 1990; Scarpaet al., Virology 180:849-52, 1991; Burns et al., Proc. Natl. Acad. Sci.USA 90:8033-37, 1993; and Boris-Lawrie and Temin, Cur. Opin. Genet.Develop. 3:102-09, 1993.

[0066] The cell line used to generate EMSP can be derived from anymammal. Particularly illustrative cell lines can be derived from human,mouse, monkey, rabbit, or pig cells. In one particular embodiment, thecell line used as a scaffold for the EMSP is a human cell line.Generally, cell lines used to generate an EMSP of the present inventionfor the generation of polyclonal T cells, expresses low or no MHC classI or class II molecules, although, cloned T cells, or T cells with knownspecificity may not require low MHC on the EMSP, as they will tend notto be allo-responsive, and simply need to be amplified in number. In oneparticular embodiment, the K562 human erythromyloid cell line is used(American Type Culture Collection, Manassas, Va.). In a furtherembodiment, the 721.221 cell line is used as a scaffold for generatingan EMSP (R. Greenwood, , Y. Shimizu, G. S. Sekhon, R. DeMars. 1994.Novel allele-specific, post-translational reduction in HLA class Isurface expression in a mutant human B cell line. J. Immunol. 153:5525;Shimizu, Y., and R. DeMars. 1989. Production of human cells expressingindividual transferred HLA-A, -B, -C genes using an HLA-A, -B, -C nullhuman cell line. J. Immunol. 142:3320). In another embodiment, the T2cell line is used as a scaffold for generating an EMSP (Salter, R. D.,J. Alexander, F. Levine, D. Pious, P. Cresswell. 1985. Evidence for twotrans-acting genes regulating HLA class II antigen expression. J.Immunol. 135:4235, III Grandea, A. G., M. J. Androlewicz, R. S. Athwal,D. E. Geraghty, T. Spies. 1995. Dependence of peptide binding by MHCclass I molecules on their interaction with TAP. Science 270:105) In yeta further embodiment, the C1R cell line is used as a scaffold forgenerating an EMSP (Edwards, P. A., C. M. Smith, A. M. Neville, M. J.O'Hare. 1982. A human-hybridoma system based on a fast-growing mutant ofthe ARH-77 plasma cell leukemia-derived line. Eur. J. Immunol. 12:641).Generally, cell lines used to generate an EMSP of the present inventionfor the generation of antigen-specific T cells, can express MHCmolecules.

[0067] In one aspect of the present invention, lipid bilayers can beused as a scaffold for generating EMSP. Illustrative lipid bilayers areas described for example in Copeland, B., and McConnel, H. M. (1980)Biochim. Biophys. Acta 599, 95-109; McMullen, T. P. W., Lewis, R. N. A.H. and McElhaney, R. N. (1994) Biophys. J. 66, 741-752; Almeida, P. F.F., et al, 1992 Biochemistry 31, 7198-7210; Tilcock, C. P. S., et al1984, Biochemistry 23, 2696-2703; Simons, K and Ikonen, E. 1997 Nature387, 569-572; Siminovitch, D. J. et al 1987, Biochim. Biophys. Acta 901,191-200. These reference are hereby incorporated by reference in theirentirety.

[0068] As discussed above, the EMSP are engineered to express a varietyof stimulatory molecules, co-stimulatory molecules, cytokines, and/orantigen. The language “nucleic acid molecule encoding such molecules” isintended to include any nucleic acid molecule that will be transcribedand translated into a protein in accordance with the present inventionupon introduction of the nucleic acid molecule into an EMSP (e.g., themolecule can further contain appropriate control elements for regulatingexpression in the EMSP). The nucleic acid molecule encoding thestimulatory, co-stimulatory and/or antigen molecules can consist of onlythe coding region of the corresponding gene, or alternatively it cancontain noncoding regions, such as 5′ or 3′ untranslated regions,introns, fragments thereof, or other sequences.

[0069] The nucleic acid molecule can encode the full length marker orco-stimulatory protein or alternatively the nucleic acid can encode apeptidic fragment thereof that is sufficient to confer enhanced cellproliferation in accordance with the present invention, when contactedwith a target cell such as a T cell. The nucleic acid can encode thenatural marker or co-stimulatory protein or fragment thereof, or amodified form of the marker or co-stimulatory protein or fragmentthereof Modified forms of the natural marker or co-stimulatory proteinthat are within the scope of the invention are described below.

[0070] The invention is intended to include the use of fragments,mutants, or variants (e.g., modified forms) of the marker, stimulatory,co-stimulatory molecule, or antigen protein that retain the ability toinduce stimulation and proliferation of T cells. A “form of the protein”is intended to mean a protein that shares a significant homology withthe natural marker, stimulatory molecule, co-stimulatory protein orantigen and is capable of effecting stimulation and proliferation of Tcells. The terms “biologically active” or “biologically active form ofthe protein,” as used herein, are meant to include forms of marker,stimulatory molecules, or co-stimulatory proteins that are capable ofeffecting enhanced activated T cell proliferation. One skilled in theart can select such forms of markers, stimulatory molecules, orco-stimulatory proteins based on their ability to enhance T cellproliferation upon introduction of a nucleic acid encoding said proteinsinto an EMSP. The ability of a specific form of marker, stimulatoryprotein, co-stimulatory protein or antigen to enhance T cellproliferation can be readily determined, for example, by measuring cellproliferation or effector function by any known assay or method,including many disclosed herein.

[0071] The nucleic acid can be a cDNA or alternatively it can be agenomic DNA fragment. Variants of the proteins described herein can beprepared b a variety of known methods, such as, for example, byintroducing nucleotide base pair modifications (e.g., substitutions,deletions, additions) to a nucleic acid molecule encoding a proteinuseful in the instant invention by standard methods, such assite-directed mutagenesis or polymerase chain reaction-mediated (PCR)mutagenesis.

[0072] Furthermore, it will be appreciated by those skilled in the artthat changes in the primary amino acid sequence of a protein useful inthe present invention are likely to be tolerated without significantlyimpairing the ability of the protein to enhance T cell proliferation.Accordingly, mutant forms of the proteins that have amino acidsubstitutions, deletions and/or additions as compared to the naturallyoccurring amino acid sequence of a comparable native protein molecule,yet still retain the functional activity of the natural form of theprotein as described herein are also encompassed by the invention. Toretain the functional properties, preferably conservative amino acidsubstitutions are made at one or more amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta.-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

[0073] To express a nucleic acid molecule encoding a desired protein,such as CD32, 4-1BBL, CD86 or other stimulatory or co-stimulatorymolecules in an EMSP, the nucleic acid must be operably linked toregulatory elements. “Operably linked” is intended to mean that thenucleotide sequence encoding the protein of interest is linked to atleast one regulatory sequence in a manner that allows expression of thenucleotide sequence in the EMSP. Regulatory sequences are selected todirect expression of the desired protein in an appropriate EMSP.Accordingly, the term “regulatory sequence” includes promoters,enhancers and other expression control elements. Such regulatorysequences are known to those skilled in the art and are furtherdescribed in Goeddel, Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990).

[0074] These regulatory elements include those required fortranscription and translation of the nucleic acid encoding themarker(s), stimulatory molecules, co-stimulatory protein(s), and antigenand may include promoters, enhancers, polyadenylation signals, and othersequences necessary for transport of the molecule to the appropriatecellular compartment, which in certain embodiments, is the outermitochondrial membrane (Gonzales-Garcia et al., Development 120:3033(1994)). When the nucleic acid is a cDNA in a recombinant expressionvector, the regulatory functions responsible for transcription and/ortranslation of the cDNA are often provided by viral sequences. Examplesof commonly used viral promoters include those derived from polyoma,adenovirus 2, cytomegalovirus and simian virus 40, and retroviral LTRs(long terminal repeats).

[0075] Regulatory sequences linked to the cDNA can be selected toprovide constitutive or inducible transcription. Inducible transcriptioncan be accomplished by, for example, use of an inducible enhancer. Thus,in a specific embodiment of the invention the nucleic acid moleculeencoding a desired protein such as CD32, CD28, 4-1BB ligand, CD86 orother stimulatory or co-stimulatory proteins is under the control of aninducible control element such that expression of the desired proteincan be turned on or off (or intermediate levels in between) using anagent which affects the inducible control element (e.g., expression canbe modulated by modulating the concentration of the inducing agent inthe presence of the T cell). This allows for switching on or off of theexpression of the protein. These regulatory sequences can functioneither in vitro or in vivo. Illustrative inducible or otherwiseregulated expression systems those controlled by heavy metals (Mayo etal. 1982 Cell 29:99-108), RU-486 (a progesterone antagonist) (Wang etal. 1994 Prxoc. Natl. Acad. Sci. USA 91:8180-8184), steroids (Mader andWhite, 1993 Proc. Natl. Acad. Sci. USA 90:5603-5607), and tetracycline(Gossen and Bujard 1992 Proc. Natl. Acad. Sci. USA 89:5547-5551; U.S.Pat. No. 5,464,758). Thus, inducible expression of a variety of proteinsuseful in the present invention can be used in vivo for production of agene therapy product. Likewise, an inducible expression system can beused to transduce or transfect a population of T cells, eitherpolyclonal, antigen-specific, clonal, or T cell lines, which can then beinfused in a patient and further induced in vivo to express a desiredprotein. In an additional embodiment, the EMSP of the present inventioncan be modified with a gene under the control of inducible expressioncontrol element. These EMSP can then also be administered to a patientand induced in vivo to express a desired protein. Within this and othercontexts of the present invention, the induced EMSP can be used to breaktolerance against tumor antigens, autoantigens, or other pathogenicantigens such as viral antigens. In another embodiment, EMSP or T cellsof the present invention can be transfected or transduced with a geneencoding a homing molecule or other so called “addressins” under thecontrol of an inducible expression element. Such modified EMSP oractivated T cells can then be induced in vivo or in vitro. Such EMSP orT cells expressing the induced gene would then be used to home to aparticular site of interest, for example a site of tumor or otherdisease such as autoimmune disease or viral infection. The skilledartisan would readily recognize that any variety of proteins would beuseful under the control of an inducible expression control element orpromoter, e.g. cytokines, homing receptors, addressing, tumor antigens,viral antigens, or other proteins useful for the recruitment of otherimmune cells to a site such as a lymph node, for destruction oractivation.

[0076] The EMSP of the present invention are generally irradiated orotherwise rendered non-dividing prior to contact with target cells suchthat the EMSP are no longer dividing. In one embodiment of the presentinvention, the EMSPs are irradiated with 100 Gy (10,000 rads). However,one of skill in the art would readily recognize that the amount ofirradiation can be optimized according to the type of EMSP. Also, otherchemical methods could serve the same function, such as formamidefixation, or mitomycin C, for example. In embodiments where synthesizedlipid bilayers are used as a scaffold for generating EMSP, the skilledartisan would recognize that treatments such as irradiation are notneccesary. Further, treatment to render the EMSP non-viable ornon-dividing is unnecessary when such cells are to be removed byselection or other means prior to infusion.

[0077] The source of EMSP can be autologous, allogeneic, syngeneic,xenogeneic, or chemically synthesized. In another embodiment, the cellscan be derived from a product of cell fusion or a cell hybrid (e.g.fusions of cells inter or intra species).

[0078] The EMSP can be stored under a variety of conditions, such as atroom temperature, 4° C., cryopreserved, or freeze-dried. The EMSPs canalso be fixed using any number of common fixatives such as formaldehydeor formamide.

[0079] The agents can be added to the EMSP before, during or aftermixing with target cells.

[0080] The EMSP of the present invention can be used in in vitro or invivo settings. For example, EMSP can be administered in vivo at localtumor sites or disease sites or can be administered systemically. Asdescribed further below, pharmaceutical compositions comprising the EMSPof the present invention are administered via any variety of routes anddoses and can be determined by the skilled artisan. EMSP can be loadedwith ligands/Abs in vivo by systemic or local administration followingadmin of EMSP. Similarly, ligand and/or ligand receptor can be inducedto be expressed following transfer using drugs, such as tetracycline, todrive expression off of inducible gene elements.

[0081] Generally, T cells of the present invention are first stimulatedresulting in upregulation and/or downregulation of certain key moleculesfollowed by or concomitant with exit from the G0 phase. Subsequently,these T cells can be expanded to large numbers using a variety ofdifferent molecules as described herein below.

[0082] Sources of T cells

[0083] In one aspect of the present invention, ex vivo T cell expansioncan be performed by isolation of T cells and subsequent stimulationfollowed by further expansion. In one embodiment of the invention, the Tcells may be stimulated by a single agent. In another embodiment, Tcells are stimulated with two agents, one that induces a primary signaland a second that is a co-stimulatory signal. Ligands useful forstimulating a single signal or stimulating a primary signal and anaccessory molecule that stimulates a second signal may be used insoluble form, attached to the surface of a cell, such as an EMSP, orimmobilized on a surface as described herein. In a preferred embodimentboth primary and secondary agents are co-immobilized on a surface, forexample a bead or an EMSP. In one embodiment, the molecule providing theprimary activation signal, such as a CD3 ligand, and the co-stimulatorymolecule, such as a CD28 ligand or 4-1BB ligand are coupled to or loadedon the same surface, for example, a particle or an EMSP. Further, asnoted earlier, one, two, or more stimulatory molecules may be used onthe same or differing surfaces or EMSP.

[0084] Prior to expansion, a source of T cells is obtained from asubject. The term “subject” is intended to include living organisms inwhich an immune response can be elicited (e.g., mammals). Examples ofsubjects include humans, dogs, cats, mice, rats, and transgenic speciesthereof. T cells can be obtained from a number of sources, includingperipheral blood mononuclear cells, bone marrow, lymph node tissue,spleen tissue, and tumors. In certain embodiments of the presentinvention, any number of T cell lines available in the art, may be used.In certain embodiments of the present invention, T cells can be obtainedfrom a unit of blood collected from a subject using any number oftechniques known to the skilled artisan, such as ficoll separation. Inone preferred embodiment, cells from the circulating blood of anindividual are obtained by apheresis or leukapheresis. The apheresisproduct typically contains lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and platelets. In one embodiment, the cells collected byapheresis may be washed to remove the plasma fraction and to place thecells in an appropriate buffer or media for subsequent processing steps.In one embodiment of the invention, the cells are washed with phosphatebuffered saline (PBS). In an alternative embodiment, the wash solutionlacks calcium and may lack magnesium or may lack many if not alldivalent cations. Again, surprisingly, initial activation steps in theabsence of calcium lead to magnified activation. As those of ordinaryskill in the art would readily appreciate a washing step may beaccomplished by methods known to those in the art, such as by using asemi-automated “flow-through” centrifuge (for example, the Cobe 2991cell processor) according to the manufacturer's instructions. Afterwashing, the cells may be resuspended in a variety of biocompatiblebuffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, theundesirable components of the apheresis sample may be removed and thecells directly resuspended in culture media.

[0085] In another embodiment, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™ gradient. A specificsubpopulation of T cells, such as CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, andCD45RO⁺T cells, can be further isolated by positive or negativeselection techniques. For example, in one preferred embodiment, T cellsare isolated by incubation with anti-CD3/anti-CD28 (i.e.,3×28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a timeperiod sufficient for positive selection of the desired T cells. In oneembodiment, the time period is about 30 minutes. In a furtherembodiment, the time period ranges from 30 minutes to 36 hours or longerand all integer values there between. In a further embodiment, the timeperiod is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferredembodiment, the time period is 10 to 24 hours. In one preferredembodiment, the incubation time period is 24 hours. For isolation of Tcells from patients with leukemia, use of longer incubation times, suchas 24 hours, can increase cell yield. Longer incubation times may beused to isolate T cells in any situation where there are few T cells ascompared to other cell types, such in isolating tumor infiltratinglymphocytes (TIL) from tumor tissue or from immunocompromisedindividuals. Further, use of longer incubation times can increase theefficiency of capture of CD8+ T cells.

[0086] Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. A preferred method iscell sorting and/or selection via negative magnetic immunoadherence orflow cytometry that uses a cocktail of monoclonal antibodies directed tocell surface markers present on the cells negatively selected. Forexample, to enrich for CD4⁺ cells by negative selection, a monoclonalantibody cocktail typically includes antibodies to CD14, CD20, CD11b,CD16, HLA-DR, and CD8.

[0087] For isolation of a desired population of cells by positive ornegative selection, the concentration of cells and surface (e.g.particles such as beads) can be varied. In certain embodiments, it maybe desirable to significantly decrease the volume in which beads andcells are mixed together (i.e., increase the concentration of cells), toensure maximum contact of cells and beads. For example, in oneembodiment, a concentration of 2 billion cells/ml is used. In oneembodiment, a concentration of 1 billion cells/ml is used. In a furtherembodiment, greater than 100 million cells/ml is used. In a furtherembodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45,or 50 million cells/ml is used. In yet another embodiment, aconcentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mlis used. In further embodiments, concentrations of 125 or 150 millioncells/ml can be used. Using high concentrations can result in increasedcell yield, cell activation, and cell expansion. Further, use of highcell concentrations allows more efficient capture of cells that mayweakly express target antigens of interest, such as CD28-negative Tcells, or from samples where there are many tumor cells present (i.e.,leukemic blood, tumor tissue, etc). Such populations of cells may havetherapeutic value and would be desirable to obtain. For example, usinghigh concentration of cells allows more efficient selection of CD8+ Tcells that normally have weaker CD28 expression.

[0088] In a related embodiment, it may be desirable to use lowerconcentrations of cells. By significantly diluting the mixture of Tcells and surface (e.g. particles such as beads), interactions betweenthe particles and cells is minimized. This selects for cells thatexpress high amounts of desired antigens to be bound to the particles.For example, CD4+ T cells express higher levels of CD28 and are moreefficiently captured than CD8+ T cells in dilute concentrations. In oneembodiment, the concentration of cells used is 5×10⁶/ml. In otherembodiments, the concentration used can be from about 1×10⁵/ml to1×10⁶/ml, and any integer value in between.

[0089] If desired or necessary, monocyte populations (i.e., CD14⁺ cells)may be depleted from blood preparations prior to ex vivo expansion by avariety of methodologies, including anti-CD14 coated beads or columns,or utilization of the phagocytotic activity of these cells to facilitateremoval. Accordingly, in one embodiment, the invention uses paramagneticparticles of a size sufficient to be engulfed by phagocytotic monocytes.In certain embodiments, the paramagnetic particles are commerciallyavailable beads, for example, those produced by Dynal AS under the tradename Dynabeads™. Exemplary Dynabeads™ in this regard are M-280, M-450,and M-500. In one aspect, other non-specific cells are removed bycoating the paramagnetic particles with “irrelevant” proteins (e.g.,serum proteins or antibodies). Irrelevant proteins and antibodiesinclude those proteins and antibodies or fragments thereof that do notspecifically target the T cells to be expanded. In certain embodimentsthe irrelevant beads include beads coated with sheep anti-mouseantibodies, goat anti-mouse antibodies, and human serum albumin.

[0090] In brief, such depletion of monocytes is performed bypreincubating PBMC isolated from whole blood or apheresed peripheralblood with one or more varieties of irrelevant or non-antibody coupledparamagnetic particles at any amount that allows for removal ofmonocytes (approximately a 20:1 bead:cell ratio) for about 30 minutes to2 hours at 22 to 37 degrees C., followed by magnetic removal of cellswhich have attached to or engulfed the paramagnetic particles. Suchseparation can be performed using standard methods available in the art.For example, any magnetic separation methodology may be used including avariety of which are commercially available, (e.g., DYNAL® MagneticParticle Concentrator (DYNAL MPC®)). Assurance of requisite depletioncan be monitored by a variety of methodologies known to those ofordinary skill in the art, including flow cytometric analysis of CD14positive cells, before and after said depletion. T cells for stimulationcan also be frozen after the washing step, which does not require themonocyte-removal step. Wishing not to be bound by theory, the freeze andsubsequent thaw step provides a more uniform product by removinggranulocytes and to some extent monocytes in the cell population. Afterthe washing step that removes plasma and platelets, the cells may besuspended in a freezing solution. While many freezing solutions andparameters are known in the art and will be useful in this context, onemethod involves using PBS containing 20% DMSO and 8% human serumalbumin, or other suitable cell freezing media, the cells then arefrozen to −80° C. at a rate of 1° per minute and stored in the vaporphase of a liquid nitrogen storage tank. Other methods of controlledfreezing may be used as well as uncontrolled freezing immediately at−20° C. or in liquid nitrogen.

[0091] Stimulation of a Cell Population

[0092] As noted above, the present invention provides compositions andmethods for stimulating a cell population by binding moieties on thesurfaces of the cells in that population. Contacting a cell populationwith an agent (e.g., a ligand) that binds to a cell surface moiety canstimulate the cell population. The ligand may be in solution but alsomay be attached to a surface. Ligation of cell surface moieties, such asa receptor, may generally induce a particular signaling pathway. Recentstudies suggest that for signaling to occur, critical concentrations oflipid rafts containing the requisite receptors must aggregate. By way ofexample, raft aggregation may be facilitated in vivo or in vitro byattaching ligands for particular cell surface moieties to an EMSP andexposing the ligand-bearing EMSP to the cells of interest.

[0093] The methods of the present invention relate to the stimulation ofa target cell by introducing a ligand or agent that binds to a cellularmoiety, thereby inducing a cellular event. Binding of the ligand oragent to the cell may trigger a signaling pathway that in turn activatesparticular phenotypic or biological changes in the cell. The stimulationof a target cell by introducing a ligand or agent that binds to acellular moiety as described herein may upregulate or downregulate anynumber of cellular processes leading to particular phenotypic orbiological changes in the cell. The activation of the cell may enhancenormal cellular functions or initiate normal cell functions in anabnormal cell. The method described herein provides stimulation bycontacting the cells with the ligand or agent that binds a cell surfacemoiety. Stimulation of a cell may be enhanced or a particular cellularevent may be stimulated by introducing a second agent or ligand thatligates a second cell surface moiety. This method may be applied to anycell for which ligation of a cell surface moiety leads to a signalingevent. The invention further provides means for selection or culturingthe stimulated cells. The prototypic example described is stimulation ofT cells, but one of ordinary skill in the art will readily appreciatethat the method may be applied to other cell types. By way of example,cell types that may be stimulated and selected include fibroblasts,neuroblasts, lung cells, hematopoietic stem cells and hematopoieticprogenitor cells (CD34⁺ cells), mesenchymal stem cells, mesenchymalprogenitor cells, neural and hepatic progenitor and stem cells,dendritic cells, cytolytic T cells (CD8⁺ cells), T helper cells (CD4+cells), B-cells, NK cells, other leukocyte populations, pluripotent stemcells, multi-potent stem cells, islet cells, etc. Accordingly, thepresent invention also provides populations of cells resulting from thismethodology as well as cell populations having distinct phenotypicalcharacteristics, including T cells with specific phenotypiccharacteristics.

[0094] As noted above a variety of cell types may be utilized within thecontext of the present invention. For example, cell types such as Bcells, T cells, NK cells, other blood cells, neuronal cells, lung cells,glandular (endocrine) cells, bone forming cells (osteoclasts, etc.),germ cells (e.g., oocytes), epithelial cells lining reproductive organs,and others may be utilized. Cell surface moiety-ligand pairs couldinclude (but not exclusively): T cell antigen receptor (TCR) andanti-CD3 mAb, TCR and major histocompatibility complex (MHC)+antigen,TCR and peptide-MHC tetramer, TCR and superantigens (e.g.,staphylococcal enterotoxin B (SEB), toxic shock syndrome toxin (TSST),etc.), B cell antigen receptor (BCR) and anti-Ig, BCR and LPS, BCR andspecific antigens (univalent or polyvalent), NK receptor and anti-NKreceptor antibodies, FAS (CD95) receptor and FAS ligand, FAS receptorand anti-FAS antibodies, CD54 and anti-CD54 antibodies, CD2 and anti-CD2antibodies, CD2 and LFA-3 (lymphocyte function related antigen-3),cytokine receptors and their respective cytokines, cytokine receptorsand anti-cytokine receptor antibodies, TNF-R (tumor necrosisfactor-receptor) family members and antibodies directed against them,TNF-R family members and their respective ligands, adhesion/homingreceptors and their ligands, adhesion/homing receptors and antibodiesagainst them, oocyte or fertilized oocyte receptors and their ligands,oocyte or fertilized oocyte receptors and antibodies against them,receptors on the endometrial lining of uterus and their ligands, hormonereceptors and their respective hormone, hormone receptors and antibodiesdirected against them, and others.

[0095] The nature of the binding of a multivalent or monovalent receptorby a ligand will either result in the multimerization of the receptors,or aggregation/orientation of the receptors, such that signaling or cellresponse is upregulated, downregulated, accelerated, improved, orotherwise altered so as to confer a particular benefit, such as celldivision, cytokine secretion, cell migration, increased cell-cellinteraction, etc.

[0096] Two examples are given below that illustrate how such amultimerization, aggregation, or controlled reorientation of cellsurface moieties could be of practical benefit.

[0097] In one example, normal T cell activation by antigen and antigenpresenting cells usually results in aggregation of TCR rafts,cytoskeletal reorganization, polarization of “activation” signals andcell division, for example. Using man-made approaches, such as thosedescribed herein, in the absence of “normal” in-vivo T cell activation,one could accelerate, improve, or otherwise affect the functionsdescribed above, in particular through the accelerated, controlled, andspatially oriented ligation of TCR and CD28 or 4-1BB or otherco-stimulatory molecules. Benefits include improved cell expansion invitro resulting in higher numbers of infuseable and more robust cellsfor therapeutic applications. Other benefits could be improved receptor“aggregation” for cells with defects, such as lower-than-normal TCRdensity on the cell surface. Similarly, in vivo applications could bebeneficial where specific T cell populations need to be activated, suchas tumor-specific T cells at tumor sites. Improved receptor aggregationand orientation could provide an activation signal otherwise difficultto obtain for functionally tolerized T cells. Within this and othercontexts of the present invention, the EMSP can be used to breaktolerance against tumor antigens, autoantigens, or other pathogenicantigens such as viral antigens. Further, such activation could be usedwithin the context of antigen specific T cells. In this regard T cellsfrom a tumor could be isolated and expanded and infused into thepatient. Similarly, T cells exposed to an antigen either in vivo or invitro could be expanded by the present methodologies.

[0098] In one particular embodiment of the invention, a T cellpopulation may be stimulated by ligating the surfaces of the T cells. Inone aspect of the present invention, antibodies to CD3 and CD28 areloaded onto an EMSP. In another aspect of the present invention, anyligand that binds the TCR/CD3 complex and initiates a primarystimulation signal may be utilized as a primary activation agent loadedonto or expressed by the EMSP. Any ligand that binds CD28 and initiatesthe CD28 signal transduction pathway, thus causing co-stimulation of thecell with a CD3 ligand and enhancing activation of a population of Tcells, is a CD28 ligand and accordingly, is a co-stimulatory agentwithin the context of the present invention.

[0099] In other aspects of the present invention, T cells can be exposedto a bead conjugated agent or soluble forms of agents or ligands priorto or concurrently with the EMSPs of the present invention as describedherein.

[0100] In certain embodiments, the EMSP of the present invention can becontacted with paramagnetic particles such that said paramagneticparticles are engulfled by the EMSP. EMSP comprising paramagneticparticles can then be subjected to magnetic force and concentrated orlocalized to a particular site of interest, such as a tumor, site ofviral infection or site of autoimmune disease, and/or otherwiseselected, either in vitro or in vivo.

The Primary Signal

[0101] The biochemical events responsible for ex vivo T cell stimulationare set forth briefly below. Interaction between the TCR/CD3 complex andantigen presented in conjunction with either MHC class I or class IImolecules on an antigen-presenting cell initiates a series ofbiochemical events termed antigen-specific T cell activation.Accordingly, activation of T cells can be accomplished by stimulatingthe T cell TCR/CD3 complex or by stimulating the CD2 surface protein. Ananti-CD3 monoclonal antibody can be used to activate a population of Tcells via the TCR/CD3 complex. A number of anti-human CD3 monoclonalantibodies are commercially available, exemplary are OKT3, prepared fromhybridoma cells obtained from the American Type Culture Collection, andmonoclonal antibody G19-4. Similarly, stimulatory forms of anti-CD2antibodies are known and available. Stimulation through CD2 withanti-CD2 antibodies is typically accomplished using a combination of atleast two different anti-CD2 antibodies. Stimulatory combinations ofanti-CD2 antibodies that have been described include the following: theT11.3 antibody in combination with the T11.1 or T11.2 antibody (Meuer etal., Cell 36:897-906, 1984), and the 9.6 antibody (which recognizes thesame epitope as T11.1) in combination with the 9-1 antibody (Yang etal., J. Immunol. 137:1097-1100, 1986). Other antibodies that bind to thesame epitopes as any of the above described antibodies can also be used.Additional antibodies, or combinations of antibodies, can be preparedand identified by standard techniques.

[0102] A primary activation signal can also be delivered to a T cellthrough other mechanisms. For example, a combination that may be usedincludes a protein kinase C (PKC) activator, such as a phorbol ester(e.g., phorbol myristate acetate), and a calcium ionophore (e.g.,ionomycin, which raises cytoplasmic calcium concentrations), or thelike. The use of such agents bypasses the TCR/CD3 complex but delivers astimulatory signal to T cells. Other agents acting as primary signalsmay include natural and synthetic ligands. A natural ligand may includeMHC with or without a peptide presented. Other ligands may include, butare not limited to, a peptide, polypeptide, growth factor, cytokine,chemokine, glycopeptide, soluble receptor, steroid, hormone, mitogen,such as PHA, or other superantigens, peptide-MHC tetramers (Altman, etal., Science. 1996 October 4;274(5284):94-6.) and soluble MHC dimers(Dal Porto, et al. Proc Natl Acad Sci USA 1993 July 15;90). Within thecontext of the present invention, the use of the EMSP for stimulationmay result in stimulation such that no secondary signal is required toinduce proliferation of T cells.

[0103] In other embodiments, signal transduction events of any kind maybe magnified or analyzed by utilizing the current invention. Forexample, G protein-coupled receptors may stimulated and measured usingthe methods of the present invention.

The Secondary Signal

[0104] While stimulation of the TCR/CD3 complex or CD2 molecule appearsto be required for delivery of a primary activation signal in a T cell,a number of molecules on the surface of T cells, termed accessory orco-stimulatory molecules, have been implicated in regulating thetransition of a resting T cell to blast transformation, and subsequentproliferation and differentiation. Thus, in addition to the primaryactivation signal, induction of T cell responses requires a second,co-stimulatory signal. One such co-stimulatory or accessory molecule,CD28, is believed to initiate or regulate a signal transduction pathwaythat is distinct from any stimulated by the TCR complex. Another suchco-stimulatory or accessory molecule, 4-1BB, preferentially stimulatesCD8+ T cells but may also be used for the stimulation of CD4+ T cells.

[0105] Therefore, to enhance activation and proliferation of apopulation of T cells in the absence of exogenous growth factors oraccessory cells, an accessory molecule on the surface of the T cell,such as CD28 or 4-1BB, is stimulated with a ligand that binds theaccessory molecule. In one embodiment, stimulation of the accessorymolecule CD28 and T cell activation occur simultaneously by contacting apopulation of T cells with an EMSP to which a ligand that binds CD3 anda ligand that binds CD28 are attached. In another embodiment,stimulation of the accessory molecule 4-1BB and T cell activation occursimultaneously by contacting a population of T cells with an EMSP towhich a ligand that binds CD3 and a ligand that binds 4-1BB areattached. Activation of the T cells, for example, with an anti-CD3antibody, and stimulation of the CD28 accessory molecule results inselective proliferation of CD4⁺ T cells. Activation of the T cells, forexample, with an anti-CD3 antibody, and stimulation of the 4-1BBaccessory molecule results in preferential proliferation of CD8⁺ Tcells.

[0106] Accordingly, one of ordinary skill in the art will recognize thatany agent, including an anti-CD28 antibody or fragment thereof capableof cross-linking the CD28 molecule, or a natural ligand for CD28 can beused to stimulate T cells. Exemplary anti-CD28 antibodies or fragmentsthereof useful in the context of the present invention includemonoclonal antibody 9.3 (IgG2_(a)) (Bristol-Myers Squibb, Princeton,N.J.), monoclonal antibody KOLT-2 (IgG1), 15E8 (IgG1), 248.23.2 (IgM),and EX5.3D10 (IgG2_(a)) (ATCC HB11373). Exemplary natural ligandsinclude the B7 family of proteins, such as B7-1 (CD80) and B7-2 (CD86)(Freedman et al., J. Immunol. 137:3260-3267, 1987; Freeman et al., J.Immunol. 143:2714-2722, 1989; Freeman et al, J. Exp. Med. 174:625-631,1991; Freeman et al., Science 262:909-911, 1993; Azuma et al., Nature366:76-79, 1993; Freeman et al., J. Exp. Med. 178:2185-2192, 1993). Inaddition, binding homologues of a natural ligand, whether native orsynthesized by chemical or recombinant techniques, can also be used inaccordance with the present invention. Other agents acting as secondarysignals may include natural and synthetic ligands. Agents may include,but are not limited to, other antibodies or fragments thereof, apeptide, polypeptide, growth factor, cytokine, chemokine, glycopeptide,soluble receptor, steroid, hormone, mitogen, such as PHA, or othersuperantigens.

[0107] Likewise, one of ordinary skill in the art will recognize thatany agent, including an anti-4-1BB antibody or fragment thereof capableof cross-linking the 4-1BB molecule, or a natural ligand for 4-1BB canbe used to stimulate T cells. In particular, human 4-1BB ligand can becloned from B cells into the pcDNA3. or other suitable vectors and betransfected into an EMSP.

[0108] In a further embodiment of the invention, activation of a T cellpopulation may be enhanced by co-stimulation of other T cell integralmembrane proteins. For example, binding of the T cell integrin LFA-1 toits natural ligand, ICAM-1, may enhance activation of cells. Anothercell surface molecule that may act as a co-stimulator for T cells isVCAM-1 (CD106) that binds very-late-antigen-4 (VLA-4) on T cells.

[0109] In certain embodiments of the present invention, stimulation,activation, and expansion of T cells using EMSP as described hereinenhances expression of certain key molecules in T cells that protectagain apoptosis or otherwise proling survival in vivo or in vitro.Apoptosis usually results from induction of a specific signal in the Tcell. Thus, the compositions and methods of the invention provide forprotecting a T cell from cell death resulting from stimulation of the Tcell. It is known in the art that presently cross-linking of the T cellreceptor, either by a polyclonal activator, such as an anti-CD3 antibodyand/or anti-CD28 antibody, or alternatively by an antigen on an antigenpresenting cell (APC), in the absence of a co-stimulatory signal, canresult in T cell anergy or T cell death. Therefore, also included in thepresent invention is the enhanced T-cell growth by protection frompremature death or from absence or depletion of recognized T cell growthmarkers, such as Bcl-xL, growth factors, cytokines, or lymphokinesnormally necessary for T cell survival, as well as from Fas or TumorNecrosis Factor Receptor (TNFR) cross-linking or by exposure to certainhormones or stress.

[0110] One of skill in the art will appreciate that cells other than Tcells may be stimulated by binding of an agent that ligates a cellsurface moiety and induces aggregation of the moiety, which in turnresults in activation of a signaling pathway. Other such cell surfacemoieties include, but are not limited to, GPI-anchored folate receptor(CD59), human IgE receptor (FcεRi receptor), BCR, EGF receptor, insulinreceptor, ephrin B1 receptor, neurotrophin, glial-cell derivedneutrophic factor (GNDF), hedgehog and other cholesterol-linked andpalmitoylated proteins, H-Ras, integrins, endothelial nitric oxidesynthase (eNOS), FAS, members of the TNF receptor family, GPI-anchoredproteins, doubly acylated proteins, such as the Src-family kinases, thealpha-subunit of heterotrimeric G proteins, and cytoskeletal proteins.

[0111] The cell population may be stimulated as described herein, suchas by contact with an anti-CD3 antibody or an anti-CD2 antibody loadedonto an EMSP engineered to express an Fcγ receptor such as CD32, or bycontact with a protein kinase C activator (e.g., bryostatin) inconjunction with a calcium ionophore. For co-stimulation of an accessorymolecule on the surface of the T cells, a ligand that binds theaccessory molecule is used. For example, a population of CD4⁺ cells canbe contacted with an anti-CD3 antibody and an anti-CD28 antibody, underconditions appropriate for stimulating proliferation of the T cells.Similarly, to stimulate proliferation of CD8⁺ T cells, an anti-CD3antibody and the 4-1BB ligand can be used. Alternatively, to stimulateproliferation of CD8⁺ T cells, an anti-CD3 antibody and the anti-CD28antibody B-T3, XR-CD28 (Diaclone, Besançon, France) can be used as canother methods commonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):1319-1328,1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0112] The primary stimulatory signal and the co-stimulatory signal forthe T cell may be provided by different protocols. For example, theagents providing each signal may be in solution or coupled to a surfacesuch as loaded on an EMSP as described herein. When loaded on an EMSP orother surface, such as a paramagnetic bead, the agents may be loaded onthe same EMSP or coupled to the same surface (i.e., in “cis” formation)or can be loaded onto separate EMSP or coupled to separate surfaces(i.e., in “trans” formation). Alternatively, one agent may be loaded onan EMSP or coupled to a surface and the other agent in solution. In oneembodiment, the agent providing the co-stimulatory signal is bound to acell surface and the agent providing the primary activation signal is insolution or coupled to a surface. In certain embodiments, both agentscan be in solution. In another embodiment, the agents may be in solubleform, and then bound to a surface, such as a cell expressing Fcreceptors or an antibody or other binding agent which will bind to theagents. In a preferred embodiment, the two agents are loaded onto thesame EMSP that has been engineered to express an FCγ receptor such asCD32. By way of example, the agent providing the primary activationsignal is an anti-CD3 antibody and the agent providing theco-stimulatory signal is an anti-CD28 antibody; and both agents areloaded onto an EMSP in equivalent molecular amounts. In one embodiment,a 1:1 ratio of each antibody loaded onto an EMSP for CD4⁺ T cellexpansion and T cell growth is used. In certain aspects of the presentinvention, a ratio of anti CD3:CD28 antibodies loaded onto an EMSP isused such that an increase in T cell expansion is observed as comparedto the expansion observed using a ratio of 1:1. In one particularembodiment an increase of from about 0.5 to about 3 fold is observed ascompared to the expansion observed using a ratio of 1:1. In oneembodiment, the ratio of CD3:CD28 antibody loaded onto an EMSP rangesfrom 100:1 to 1:1000 and all integer values there between. In one aspectof the present invention, more anti-CD28 antibody is loaded onto an EMSPthan anti-CD3 antibody, i.e. the ratio of CD3:CD28 is less than one. Incertain embodiments of the invention, the ratio of anti CD28 antibody toanti CD3 antibody loaded onto an EMSP is greater than 2:1. In oneparticular embodiment, a 1:1000 CD3:CD28 ratio of antibody loaded ontoan EMSP is used. In another embodiment, a 1:500 CD3:CD28 ratio ofantibody loaded onto an EMSP is used. In another embodiment, a 1:250CD3:CD28 ratio of antibody loaded onto an EMSP is used. In anotherembodiment, a 1:100 CD3:CD28 ratio of antibody loaded onto an EMSP isused. In a further embodiment, a 1:50 CD3:CD28 ratio of antibody loadedonto an EMSP is used. In another embodiment, a 1:30 CD3:CD28 ratio ofantibody loaded onto an EMSP is used. In one preferred embodiment, a1:100 CD3:CD28 ratio of antibody loaded onto an EMSP is used. In anotherpreferred embodiment, a 1:50 CD3:CD28 ratio of antibody loaded onto anEMSP is used. In another preferred embodiment, a 1:25 CD3:CD28 ratio ofantibody loaded onto an EMSP is used. In one preferred embodiment, a1:10 CD3:CD28 ratio of antibody loaded onto an EMSP is used. In anotherembodiment, a 1:3 CD3:CD28 ratio of antibody loaded onto an EMSP isused. In yet another embodiment, a 3:1 CD3:CD28 ratio of antibody loadedonto an EMSP is used.

[0113] Ratios of EMSP, or other particles as described herein, to cellsfrom 1:10000 to 10000:1 and any integer values in between may be used tostimulate T cells or other target cells. As those of ordinary skill inthe art can readily appreciate, the ratio of EMSP to cells may dependanton EMSP size relative to the target cell. For example, a small EMSPcould only bind a few cells, while a large EMSP could bind many. Incertain embodiments the ratio of cells to EMSP ranges from 1:100 to100:1 and any integer values in-between and in further embodiments theratio comprises 1:9 to 9:1 and any integer values in between, can alsobe used to stimulate T cells. In a preferred embodiment wherein the EMSPis K32 as described herein in the Examples, the ratio of target cell toEMSP is most suitable at about 1:1 to about 1:100. The ratio ofanti-CD3- and anti-CD28-loaded EMSP to T cells that result in T cellstimulation can vary as noted above, however certain preferred valuesinclude at least 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1 to 6:1, with onepreferred ratio being at least 1:1 EMSP per T cell. In one embodiment, aratio of EMSP to cells of 1:1 or less is used.

[0114] Using certain methodologies it may be advantageous to maintainlong-term stimulation of a population of T cells following the initialactivation and stimulation, by separating the T cells from the stimulusafter a period of about 12 to about 14 days. The rate of T cellproliferation is monitored periodically (e.g., daily) by, for example,examining the size or measuring the volume of the T cells, such as witha Coulter Counter. In this regard, a resting T cell has a mean diameterof about 6.8 microns, and upon initial activation and stimulation, inthe presence of the stimulating ligand, the T cell mean diameter willincrease to over 12 microns by day 4 and begin to decrease by about day6. When the mean T cell diameter decreases to approximately 8 microns,the T cells may be reactivated and re-stimulated to induce furtherproliferation of the T cells. Alternatively, the rate of T cellproliferation and time for T cell re-stimulation can be monitored byassaying for the presence of cell surface molecules, such as, CD154,CD54, CD25, CD137, CD134, which are induced on activated T cells.Intracellular or secreted cytokines can also be monitored such as IL-2,IFN-γ, TNF-α, GM-CSF, etc.

[0115] In further embodiments of the present invention, the cells, suchas T cells, are combined with loaded EMSP, the EMSP and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the antibody or ligand loadedEMSP and cells are not separated but are cultured together.

[0116] By way of example, when T cells are the target cell population,the cell surface moieties may be ligated by allowing irradiated EMSPexpressing CD86 and to which anti-CD3 and anti-CD28 are attached via theFcγ receptor to contact the T cells. In another example of the presentinvention, when T cells are the target cell population, the cell surfacemoieties may be ligated by allowing irradiated EMSP expressing the 4-1BBligand and to which anti-CD3 and anti-CD28 are attached via the Fcγreceptor to contact the T cells.

[0117] The buffer that the cells are suspended in may be any that isappropriate for the particular cell type. When utilizing certain celltypes the buffer may contain other components, e.g. 1-5% serum,necessary to maintain cell integrity during the process. In anotherembodiment, the cells and EMSP may be combined in cell culture media.The cells and EMSP may be mixed, for example, by rotation, agitation orany means for mixing, for a period of time ranging from one minute toseveral hours. As noted above, generally the EMSP of the presentinvention are irradiated prior to contact with target cells such as Tcells. Generally, EMSP are irradiated prior to being loaded withantibodies or ligands as described herein.

[0118] In one embodiment of the present invention, the mixture may becultured for several hours (about 3 hours) to about 20 days or anyhourly integer value in between. In another embodiment, the mixture maybe cultured for 21 days. In one embodiment of the invention the EMSP andthe T cells are cultured together for about eight days. In anotherembodiment, the EMSP and T cells are cultured together for 2-3 days.Several cycles of stimulation may also be desired such that culture timeof T cells can be 60 days or more. Conditions appropriate for T cellculture include an appropriate media (e.g., Minimal Essential Media orRPMI Media 1640 or, X-vivo 15, (BioWhittaker)) that may contain factorsnecessary for proliferation and viability, including serum (e.g., fetalbovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4,GM-CSF, IL-10, IL-12, TGFβ, and TNF-α. or any other additives for thegrowth of cells known to the skilled artisan. Other additives for thegrowth of cells include, but are not limited to, surfactant, plasmanate,and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol.Media can include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15,and X-Vivo 20, with added amino acids and vitamins, either serum-free orsupplemented with an appropriate amount of serum (or plasma) or adefined set of hormones, and/or an amount of cytokine(s) sufficient forthe growth and expansion of T cells. Antibiotics, e.g., penicillin andstreptomycin, are included only in experimental cultures, not incultures of cells that are to be infused into a subject. The targetcells are maintained under conditions necessary to support growth, forexample, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g.,air plus 5% CO₂).

[0119] Cells stimulated by the methods of the present invention areactivated as shown by the induction of signal transduction, expressionof cell surface markers and/or proliferation. One such markerappropriate for T cells is CD154 which is an important immunomodulatingmolecule. The expression of CD154 is extremely beneficial in amplifyingthe immune response. CD154 interacts with the CD40 molecule expressed onmany B cells, dendritic cells, monocytes, and some endothelial cells.Accordingly, this unexpected and surprising increase in CD154 expressionis likely to lead to more efficacious T cell compositions. Stimulationof CD3⁺ cells as described herein provides T cells that express a 1.1 to20-fold increases in the levels of certain cell surface markers such asCD154 expression on days 1, 2, 3, or 4 following stimulation. Expressionof another cell surface marker, CD25, also was greater on T cells afterstimulation than on cells prior to culture or cells stimulated by othermethods. Further, after simulation using the methods of the presentinvention, CD8+ T cells show increased Bcl-xL and IL-2 expression asdescribed in the Examples below.

[0120] In another embodiment, T cells are initially stimulated with theEMSP and the T cells are then purified using any number of methodsdescribed herein, also including density gradient separation,elutriation and removal of dead cells. The remaining T cell populationis substantially free of non-T cells and can then be incubated with anynumber of cytokines such as IL-2 in culture medium in order to promotefurther T cell proliferation.

[0121] One of skill in the art will appreciate that any target cell thatcan be stimulated by cell surface moiety ligation may be combined withthe EMSP. Further, the EMSP may be separated from the cells prior toculture, at any point during culture, or at the termination of culture.In addition, the EMSP ligated to the target cells may be separated fromthe non-binding cells prior to culture or the other cells may remain inculture as well. In one embodiment, prior to culture, the EMSP andtarget cells are not separated but are cultured together.

[0122] Also contemplated by this invention, are means to increase theconcentration of the target cells, for example, a T cell fraction boundto an EMSP coated with primary and secondary stimulatory molecules. Forexample, forces greater than gravitational force may be applied, forexample, but not limited to, centrifugal force, transmembrane pressure,and a hydraulic force. Concentration may also be accomplished byfiltration. In certain embodiments, the EMSP of the present inventioncan be contacted with paramagnetic particles such that said paramagneticparticles are engulfed by the EMSP. EMSP comprising paramagneticparticles can then be subjected to magnetic force and concentratedand/or otherwise selected, either in vitro or in vivo.

[0123] One of skill in the art will readily appreciate that contactbetween the agent-coated EMSP and the cells to be stimulated can beincreased by concentration using other forces. Accordingly, any meansfor concentrating cells with cell surface moiety binding ligands will besufficient as long as the concentration brings together cells and agentsin a manner that exceeds gravity or diffusion.

[0124] A cellular event induced by contact of target cells with the EMSPof the present invention may include, for example, receptor-mediatedsignal transduction that induces or suppresses a particular pathway,including an apoptotic pathway, or induces phosphorylation of proteins,or stimulates or suppresses growth signals.

[0125] In another embodiment, the time of exposure to stimulatory agentssuch as anti-CD3/anti-CD28-coated EMSP expressing CD86 or 4-1BB may bemodified or tailored to obtain a desired T cell phenotype.Alternatively, a desired population of T cells can be selected using anynumber of selection techniques, prior to stimulation. One may desire agreater population of CD4+ T cells as opposed to CD8⁺ or regulatory Tcells, because an expansion of CD4+ T cells could improve or restoreoverall immune responsiveness. While many specific immune responses aremediated by CD8⁺ antigen-specific T cells, which can directly lyse orkill undesired cells, most immune responses require the help of CD4⁺ Tcells, which express important immune-regulatory molecules, such asGM-CSF, CD40L, and IL-2, for example. Increased numbers of CD4⁺ T cellscan increase the amount of cell-expressed CD40L introduced intopatients, potentially improving target cell visibility (improved APCfunction). Similar effects can be seen by increasing the number ofinfused cells expressing GM-CSF, or IL-2, all of which are expressedpredominantly by CD4⁺ T cells. Alternatively, in situations whereCD4-help is needed less and increased numbers of CD8⁺ T cells aredesirous, the approaches described herein can also be utilized, by forexample, pre-selecting for CD8⁺ cells prior to stimulation and/orculture. Such situations may exist where increased levels of IFN-γ orincreased cytolysis of an undesired cell is preferred. In certain otherembodiments, selection of a CD28-negative population may be desired.

[0126] T cells that have been exposed to varied stimulation times andEMSP expressing a variety of molecules may exhibit differentcharacteristics. For example, typical blood or apheresed peripheralblood mononuclear cell products have a CD4+ T cell population that isgreater than the cytotoxic or suppressor T cell population (T_(C),CD8⁺). Ex vivo expansion of T cells by stimulating CD3 and CD28receptors produces a population of T cells that prior to about days 8-9consists predominately of T_(H) cells, while after about days 8-9, thepopulation of T cells comprises an increasingly greater population ofT_(C) cells. Furthermore, one aspect of the present invention is thefinding that stimulation with an EMSP expressing 4-1BB ligand and coatedwith anti-CD3 and CD28 antibodies preferentially stimulates and expandsCD8+ cytotoxic T cells. Accordingly, depending on the purpose oftreatment, infusing a subject with a T cell population comprisingpredominately of T_(H) cells expanded with anti-CD3/anti-CD28 and CD86EMSP may be advantageous. Similarly, if an antigen-specific subset ofT_(C) cells has been isolated it may be beneficial to expand this subsetto a greater degree using EMSP expressing 4-1BB and further coated withanti-CD3 and anti-CD28 antibodies.

[0127] Further, in addition to CD4 and CD8 markers, other phenotypicmarkers vary significantly, but in large part, reproducibly during thecourse of the cell expansion process. Thus, such reproducibility enablesthe ability to tailor an activated T cell product for specific purposes.

[0128] In one such example, among the important phenotypic markers thatreproducibly vary with time are the high affinity IL-2 receptor (CD25),CD40 ligand (CD154), and CD45RO (a molecule that by preferentialassociation with the TCR may increase the sensitivity of the TCR toantigen binding). As one of ordinary skill in the art readilyappreciates, such molecules are important for a variety of reasons. Forexample, CD25 constitutes an important part of the autocrine loop thatallows rapid T cell division. CD154 has been shown to play a key role instimulating maturation of the antigen-presenting dendritic cells;activating B-cells for antibody production; regulating T_(H) cellproliferation; enhancing T_(C) cell differentiation; regulating cytokinesecretion of both T_(H) cells and antigen-presenting cells; andstimulating expression of co-stimulatory ligands, including CD80, CD86,and CD154.

[0129] Cytokine production peaks in the first few days of the ex vivoexpansion process. Accordingly, because cytokines are known to beimportant for mediating T cell activation and function as well as immuneresponse modulation, such cytokines are likely critical in thedevelopment of a therapeutic T cell product, that is able to undergoreactivation upon contact with an additional antigen challenge.Cytokines important in this regard, include, but are not limited to,IL-2, IL-4, TNF-α, and IFN-γ. Thus, by obtaining a population of T cellsduring the first few days of expansion and infusing these cells into asubject, a therapeutic benefit may occur in which additional activationand expansion of T cells in vivo occurs.

[0130] In addition to the cytokines and the markers discussedpreviously, expression of adhesion molecules known to be important formediation of T cell activation and immune response modulation alsochange dramatically but reproducibly over the course of the ex vivoexpansion process. For example, CD62L is important for homing of T cellsto lymphoid tissues and trafficking T cells to sites of inflammation.Under certain circumstances of disease and injury, the presence ofactivated T cells at these sites may be disadvantageous. Becausedown-regulation of CD62L occurs early following activation, the T cellscould be expanded for shorter periods of time. Conversely, longerperiods of time in culture would generate a T cell population withhigher levels of CD62L and thus a higher ability to target the activatedT cells to these sites under other preferred conditions. Another exampleof a polypeptide whose expression varies over time is CD49d, an adhesionmolecule that is involved in trafficking lymphocytes from blood totissues spaces at sites of inflammation. Binding of the CD49d ligand toCD49d also allows the T cell to receive co-stimulatory signals foractivation and proliferation through binding by VCAM-1 or fibronectinligands. The expression of the adhesion molecule CD54, involved in Tcell-APC and T cell-T cell interactions as well as homing to sites ofinflammation, also changes over the course of expansion. Accordingly, Tcells could be stimulated for selected periods of time that coincidewith the marker profile of interest and subsequently collected andinfused. Thus, T cell populations could be tailored to express themarkers believed to provide the most therapeutic benefit for theindication to be treated.

[0131] Those of ordinary skill in the art will readily appreciate thatthe cell stimulation methodologies described herein may be carried outin a variety of environments (i.e., containers). For example, suchcontainers may be culture flasks, culture bags, or any container capableof holding cells, preferably in a sterile environment. In one embodimentof the present invention a bioreactor is also useful. For example,several manufacturers currently make devices that can be used to growcells and be used in combination with the methods of the presentinvention. See for example, Celdyne Corp., Houston, Tex.; UnisynTechnologies, Hopkinton, Mass.; Synthecon, Inc. Houston, Tex.; AastromBiosciences, Inc. Ann Arbor, Mich.; Wave Biotech LLC, Bedminster, N.J.Further, patents covering such bioreactors include U.S. Pat. Nos.6,096,532; 5,985,653; 5,888,807; 5,190,878, which are incorporatedherein by reference.

[0132] One aspect of the present invention provides culturing cells in arocking, closed system and results in a profound enhancement inactivation and expansion of these cells. Accordingly, in one embodiment,a bioreactor with a base rocker platform is used, for example such as“THE WAVE BIOREACTOR™” (Wave Biotech LLC, Bedminster, N.J.), that allowsfor varying rates of rocking and at a variety of different rockingangles. The skilled artisan will recognize that any platform that allowsfor the appropriate motion for optimal expansion of the cells is withinthe context of the present invention.

[0133] In certain embodiments, the capacity of the bioreactor containerranges from about 0.1 liter to about 200 liters of medium. The skilledartisan will readily appreciate that the volume used for culture willvary depending on the number of starting cells and on the final numberof cells desired. In a related embodiment, the entire process ofstimulation, activation, and expansion takes place using staticconditions and/or in a bioreactor. Illustrative bioreactors include, butare not limited to, “THE WAVE BIOREACTOR™”.

[0134] In one particular embodiment, the cell stimulation methods of thepresent invention are carried out in a closed system, such as abioreactor, that allows for perfusion of medium at varying rates, suchas from about 0.1 ml/minute to about 3 ml/minute. Accordingly, incertain embodiments, the container of such a closed system comprises anoutlet filter, an inlet filter, and a sampling port for sterile transferto and from the closed system. In other embodiments, the container ofsuch a closed system comprises a syringe pump and control for steriletransfer to and from the closed system. Further embodiments provide fora mechanism, such as a load cell, for controlling media in-put andout-put by continuous monitoring of the weight of the bioreactorcontainer. In one embodiment the system comprises a gas manifold. Inanother embodiment, the bioreactor of the present invention comprises aCO₂ gas mix rack that supplies a mixture of ambient air and CO₂ to thebioreactor container and maintains the container at positive pressure.In another embodiment, the bioreactor of the present invention comprisesa variable heating element.

[0135] In one embodiment, media is allowed to enter the containerstarting on day 2, 3, 4, 5, or 6 at about 0.5 to 5.0 liters per dayuntil the desired final volume is achieved. In one preferred embodiment,media enters the container at 2 liters per day starting at day 4, untilthe volume reaches 10 liters. Once desired volume is achieved, perfusionof media can be initiated. In certain embodiments, perfusion of mediathrough the system is initiated on about day 2, 3, 4, 5, 6, 7, 8, 9, 10,11, or 12 of culture. In one embodiment, perfusion is initiated when thevolume is at about 0.1 liter to about 200 liters of media. In oneparticular embodiment, perfusion is initiated when the final volume isat 4, 5, 6, 7, 8, 9, 10, or 20 liters.

[0136] In a further embodiment of the present invention, the cells, suchas T cells, are cultured for up to 5 days in a closed, static system andthen transferred to a closed system that comprises a rocking element toallow rocking of the culture container at varying speeds.

[0137] Although the antibodies used in the methods described herein canbe readily obtained from public sources, such as the ATCC, antibodies toT cell accessory molecules and the CD3 complex can be produced bystandard techniques. Methodologies for generating antibodies for use inthe methods of the invention are well-known in the art and are discussedin further detail herein.

[0138] Agents

[0139] Agents contemplated by the present invention include proteinligands, natural ligands, and synthetic ligands. Agents that can bind tocell surface moieties, and under certain conditions, cause ligation andaggregation that leads to signaling include, but are not limited to,lectins (for example, PHA, lentil lectins, concanavalin A), antibodies,antibody fragments, peptides, polypeptides, glycopeptides, receptors, Bcell receptor and T cell receptor ligands, extracellular matrixcomponents, steroids, hormones (for example, growth hormone,corticosteroids, prostaglandins, tetra-iodo thyronine), bacterialmoieties (such as lipopolysaccharides), mitogens, antigens,superantigens and their derivatives, growth factors, cytokine, viralproteins (for example, HIV gp-120), adhesion molecules (such as,L-selectin, LFA-3, CD54, LFA-1), chemokines, and small molecules. Theagents may be isolated from natural sources such as cells, bloodproducts, and tissues, or isolated from cells propagated in vitro, orprepared recombinantly, or by other methods known to those with skill inthe art.

[0140] In one aspect of the present invention, when it is desirous tostimulate T cells, useful agents include ligands that are capable ofbinding the CD3/TCR complex, CD2, and/or CD28, and/or 4-1BB andinitiating activation or proliferation, respectively. Accordingly, theterm ligand includes those proteins that are the “natural” ligand forthe cell surface protein, such as a B7 molecule for CD28, as well asartificial ligands such as antibodies directed to the cell surfaceprotein or fusions of antibodies or other ligands. Such antibodies andfragments thereof may be produced in accordance with conventionaltechniques, such as hybridoma methods and recombinant DNA and proteinexpression techniques. Useful antibodies and fragments may be derivedfrom any species, including humans, or may be formed as chimericproteins, which employ sequences from more than one species.

[0141] Methods well known in the art may be used to generate antibodies,polyclonal antisera, or monoclonal antibodies that are specific for aligand. Antibodies also may be produced as genetically engineeredimmunoglobulins (Ig) or Ig fragments designed to have desirableproperties. For example, by way of illustration and not limitation,antibodies may include a recombinant IgG that is a chimeric fusionprotein having at least one variable (V) region domain from a firstmammalian species and at least one constant region domain from a seconddistinct mammalian species. Most commonly, a chimeric antibody hasmurine variable region sequences and human constant region sequences.Such a murine/human chimeric immunoglobulin may be “humanized” bygrafting the complementarity determining regions (CDRs), which conferbinding specificity for an antigen, derived from a murine antibody intohuman-derived V region framework regions and human-derived constantregions. Fragments of these molecules may be generated by proteolyticdigestion, or optionally, by proteolytic digestion followed by mildreduction of disulfide bonds and alkylation, or by recombinant geneticengineering techniques.

[0142] Antibodies are defined to be “immunospecific” if theyspecifically bind the ligand with an affinity constant, K_(a,) ofgreater than or equal to about 10⁴ M⁻¹, preferably of greater than orequal to about 10⁵ M⁻¹, more preferably of greater than or equal toabout 10⁶ M⁻¹, and still more preferably of greater than or equal toabout 10⁷ M⁻¹. Affinities of binding partners or antibodies can bereadily determined using conventional techniques, for example, thosedescribed by Scatchard et al. (Ann. N.Y. Acad. Sci. USA 51:660, 1949) orby surface plasmon resonance (BIAcore, Biosensor, Piscataway, N.J.) See,e.g., Wolff et al., Cancer Res., 53:2560-2565, 1993).

[0143] Antibodies may generally be prepared by any of a variety oftechniques known to those having ordinary skill in the art (See, e.g.,Harlow et al., Antibodies: A Laboratory Manual, 1988, Cold Spring HarborLaboratory). In one such technique, an animal is immunized with theligand as antigen to generate polyclonal antisera. Suitable animalsinclude rabbits, sheep, goats, pigs, cattle, and may include smallermammalian species, such as, mice, rats, and hamsters.

[0144] An immunogen may be comprised of cells expressing the ligand,purified or partially purified ligand polypeptides or variants orfragments thereof, or ligand peptides. Ligand peptides may be generatedby proteolytic cleavage or may be chemically synthesized. Peptides forimmunization may be selected by analyzing the primary, secondary, ortertiary structure of the ligand according to methods know to thoseskilled in the art in order to determine amino acid sequences morelikely to generate an antigenic response in a host animal (See, e.g.,Novotny, Mol. Immunol. 28:201-207, 1991; Berzoksky, Science 229:932-40,1985).

[0145] Preparation of the immunogen may include covalent coupling of theligand polypeptide or variant or fragment thereof, or peptide to anotherimmunogenic protein, such as, keyhole limpet hemocyanin or bovine serumalbumin. In addition, the peptide, polypeptide, or cells may beemulsified in an adjuvant (See Harlow et al., Antibodies: A LaboratoryManual, 1988 Cold Spring Harbor Laboratory). In general, after the firstinjection, animals receive one or more booster immunizations accordingto a preferable schedule for the animal species. The immune response maybe monitored by periodically bleeding the animal, separating the sera,and analyzing the sera in an immunoassay, such as an Ouchterlony assay,to assess the specific antibody titer. Once an antibody titer isestablished, the animals may be bled periodically to accumulate thepolyclonal antisera. Polyclonal antibodies that bind specifically to theligand polypeptide or peptide may then be purified from such antisera,for example, by affinity chromatography using protein A or using theligand polypeptide or peptide coupled to a suitable solid support.

[0146] Monoclonal antibodies that specifically bind ligand polypeptidesor fragments or variants thereof may be prepared, for example, using thetechnique of Kohler and Milstein (Nature, 256:495-497, 1975; Eur. J.Immunol. 6:511-519, 1976) and improvements thereto. Hybridomas, whichare immortal eucaryotic cell lines, may be generated that produceantibodies having the desired specificity to a the ligand polypeptide orvariant or fragment thereof. An animal—for example, a rat, hamster, orpreferably mouse—is immunized with the ligand immunogen prepared asdescribed above. Lymphoid cells, most commonly, spleen cells, obtainedfrom an immunized animal may be immortalized by fusion with adrug-sensitized myeloma cell fusion partner, preferably one that issyngeneic with the immunized animal. The spleen cells and myeloma cellsmay be combined for a few minutes with a membrane fusion-promotingagent, such as polyethylene glycol or a nonionic detergent, and thenplated at low density on a selective medium that supports the growth ofhybridoma cells, but not myeloma cells. A preferred selection media isHAT (hypoxanthine, aminopterin, thymidine). After a sufficient time,usually about 1 to 2 weeks, colonies of cells are observed. Singlecolonies are isolated, and antibodies produced by the cells may betested for binding activity to the ligand polypeptide or variant orfragment thereof. Hybridomas producing antibody with high affinity andspecificity for the ligand antigen are preferred. Hybridomas thatproduce monoclonal antibodies that specifically bind to a ligandpolypeptide or variant or fragment thereof are contemplated by thepresent invention.

[0147] Monoclonal antibodies may be isolated from the supernatants ofhybridoma cultures. An alternative method for production of a murinemonoclonal antibody is to inject the hybridoma cells into the peritonealcavity of a syngeneic mouse. The mouse produces ascites fluid containingthe monoclonal antibody. Contaminants may be removed from the antibodyby conventional techniques, such as chromatography, gel filtration,precipitation, or extraction.

[0148] Human monoclonal antibodies may be generated by any number oftechniques. Methods include but are not limited to, Epstein Barr Virus(EBV) transformation of human peripheral blood cells (see, U.S. Pat. No.4,464,456), in vitro immunization of human B cells (see, e.g., Boerneret al., J. Immunol. 147:86-95, 1991), fusion of spleen cells fromimmunized transgenic mice carrying human immunoglobulin genes and fusionof spleen cells from immunized transgenic mice carrying immunoglobulingenes inserted by yeast artificial chromosome (YAC) (see, e.g., U.S.Pat. No. 5,877,397; Bruggemann et al., Curr. Opin. Biotechnol. 8:455-58,1997; Jakobovits et al., Ann. N.Y. Acad. Sci. 764:525-35, 1995), orisolation from human immunoglobulin V region phage libraries.

[0149] Chimeric antibodies and humanized antibodies for use in thepresent invention may be generated. A chimeric antibody has at least oneconstant region domain derived from a first mammalian species and atleast one variable region domain derived from a second distinctmammalian species (See, e.g., Morrison et al., Proc. Natl. Acad. Sci.USA, 81:6851-55, 1984). Most commonly, a chimeric antibody may beconstructed by cloning the polynucleotide sequences that encode at leastone variable region domain derived from a non-human monoclonal antibody,such as the variable region derived from a murine, rat, or hamstermonoclonal antibody, into a vector containing sequences that encode atleast one human constant region. (See, e.g., Shin et al., MethodsEnzymol. 178:459-76, 1989; Walls et al., Nucleic Acids Res. 21:2921-29,1993). The human constant region chosen may depend upon the effectorfunctions desired for the particular antibody. Another method known inthe art for generating chimeric antibodies is homologous recombination(U.S. Pat. No. 5,482,856). Preferably, the vectors will be transfectedinto eukaryotic cells for stable expression of the chimeric antibody.

[0150] A non-human/human chimeric antibody may be further geneticallyengineered to create a “humanized” antibody. Such an antibody has aplurality of CDRs derived from an immunoglobulin of a non-humanmammalian species, at least one human variable framework region, and atleast one human immunoglobulin constant region. Humanization may yieldan antibody that has decreased binding affinity when compared with thenon-human monoclonal antibody or the chimeric antibody. Those havingskill in the art, therefore, use one or more strategies to designhumanized antibodies.

[0151] Within certain embodiments, the use of antigen-binding fragmentsof antibodies may be preferred. Such fragments include Fab fragments orF(ab′)₂ fragments, which may be prepared by proteolytic digestion withpapain or pepsin, respectively. The antigen binding fragments may beseparated from the Fc fragments by affinity chromatography, for example,using immobilized protein A or immobilized ligand polypeptide or avariant or a fragment thereof. An alternative method to generate Fabfragments includes mild reduction of F(ab′)₂ fragments followed byalkylation (See, e.g., Weir, Handbook of Experimental Immunology, 1986,Blackwell Scientific, Boston).

[0152] Non-human, human, or humanized heavy chain and light chainvariable regions of any of the above described Ig molecules may beconstructed as single chain Fv (sFv) fragments (single chainantibodies). See, e.g., Bird et al., Science 242:423-426, 1988; Hustonet al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988. Multi-functionalfusion proteins may be generated by linking polynucleotide sequencesencoding an sFv in-frame with polynucleotide sequences encoding variouseffector proteins. These methods are known in the art, and aredisclosed, for example, in EP-B1-0318554, U.S. Pat. No. 5,132,405, U.S.Pat. No. 5,091,513, and U.S. Pat. No. 5,476,786.

[0153] An additional method for selecting antibodies that specificallybind to a ligand polypeptide or variant or fragment thereof is by phagedisplay (See, e.g., Winter et al., Annul. Rev. Immunol. 12:433-55, 1994;Burton et al., Adv. Immunol. 57:191-280, 1994). Human or murineimmunoglobulin variable region gene combinatorial libraries may becreated in phage vectors that can be screened to select Ig fragments(Fab, Fv, sFv, or multimers thereof) that bind specifically to a ligandpolypeptide or variant or fragment thereof (See, e.g., U.S. Pat. No.5,223,409; Huse et al., Science 246:1275-81, 1989; Kang et al., Proc.Natl. Acad. Sci. USA 88:4363-66, 1991; Hoogenboom et al., J. Molec.Biol. 227:381-388, 1992; Schlebusch et al., Hybridoma 16:47-52, 1997 andreferences cited therein).

[0154] In certain aspects of the present invention other agents can beused in the generation of EMSP, including but not limited to fusionproteins comprising natural ligands that bind to T cell surfacemolecules. -In one embodiment, fusion proteins can be generated suchthat Ig Fe portions are fused to a natural ligand of interest. Such afusion protein could then be loaded onto an EMSP as described hereinthat expresses an Fcγ receptor.

[0155] Cell Populations

[0156] As discussed above, the present invention has broad applicabilityto any cell type having a cell surface moiety that one is desirous ofligating. In this regard, many cell signaling events can be enhanced bythe methods of the present invention. Such methodologies can be usedtherapeutically in an ex vivo setting to activate and stimulate cellsfor infusion into a patient or could be used in vivo, to induce cellsignaling events on a target cell population. However, as also notedabove, the prototypic example provided herein is directed to T cells,but is in no way limited thereto.

[0157] With respect to T cells, the T cell populations resulting fromthe various expansion methodologies described herein may have a varietyof specific phenotypic properties, depending on the conditions employed.Such phenotypic properties include enhanced expression of CD25, CD154,IFN-γ and GM-CSF, as well as altered expression of CD137, CD134, CD62L,and CD49d. The ability to differentially control the expression of thesemoieties may be very important. For example, higher levels of surfaceexpression of CD154 on “tailored T cells,” through contact with CD40molecules expressed on antigen-presenting cells (such as dendriticcells, monocytes, and even leukemic B cells or lymphomas), will enhanceantigen presentation and immune function. Such strategies are currentlybeing employed by various companies to ligate CD40 via antibodies orrecombinant CD40L. The approach described herein permits this samesignal to be delivered in a more physiological manner, e.g., by the Tcell. The ability to increase IFN-γ secretion by tailoring the T cellactivation process could help promote the generation of TH1-type immuneresponses, important for anti-tumor and anti-viral responses. LikeCD154, increased expression of GM-CSF can serve to enhance APC function,particularly through its effect on promoting the maturation of APCprogenitors into more functionally competent APC, such as dendriticcells. Altering the expression of CD137 and CD134 can effect a T cell'sability to resist or be susceptible to apoptotic signals. Controllingthe expression of adhesion/homing receptors, such as CD62L and/or CD49dmay determine the ability of infused T cells to home to lymphoid organs,sites of infection, or tumor sites.

[0158] An additional aspect of the present invention provides a T cellpopulation or composition that has been depleted of CD8⁺ or CD4⁺ cellsprior to expansion. In one embodiment, CD8⁺ cells are depleted byantibodies directed to the CD8⁺ marker. One of ordinary skill in the artwould readily be able to identify a variety of particular methodologiesfor depleting a sample of CD8⁺ or CD4⁺ cells or conversely enriching theCD4⁺ or CD8⁺ cell content. With respect to enriching for CD4⁺ cells, oneaspect of the present invention is focused on the identification of anextremely robust CD154 expression profile upon stimulation of T cellpopulations wherein T_(C) (CD8⁺) cells have been depleted. As indicatedabove, CD154 is an important immunomodulating molecule whose expressionis extremely beneficial in amplifying the immune response. Accordinglyan increase in CD154 expression is likely to lead to more efficacious Tcell compositions.

[0159] An additional aspect of the present invention provides a T cellpopulation or composition that has been depleted or enriched forpopulations of cells expressing a variety of markers, such as CD62L,CD45RA or CD45RO, cytokines (e.g. IL-2, IFN-γ, IL-4, IL-10), cytokinereceptors (e.g. CD25), perform, adhesion molecules (e.g. VLA-1, VLA-2,VLA-4, LPAM-1, LFA-1), and/or homing molecules (e.g. L-Selectin), priorto expansion. In one embodiment, cells expressing any of these markersare depleted or positively selected by antibodies or otherligands/binding agents directed to the marker. One of ordinary skill inthe art would readily be able to identify a variety of particularmethodologies for depleting or positively selecting for a sample ofcells expressing a desired marker.

[0160] The phenotypic properties of T cell populations of the presentinvention can be monitored by a variety of methods including standardflow cytometry methods and ELISA methods known by those skilled in theart.

[0161] Methods of Use

[0162] In addition to the methods described above, the EMSP describedherein and the cells stimulated and/or activated by the methods hereindescribed may be utilized in a variety of contexts. With respect to theprototypic example of T cells, the methodologies described herein can beused to selectively activate and expand a population expressing any oneor more of CD28, CD4, CD8, Bcl-xL, CD45RA, or CD45RO for use in thetreatment of infectious diseases, cancer, and immunotherapy. As aresult, a phenotypically unique population of T cells, which ispolyclonal with respect to antigen reactivity, but essentiallyhomogeneous with respect to either CD4⁺ or CD8⁺ can be produced. Inaddition, the method allows for the expansion of a population of T cellsin numbers sufficient to reconstitute an individual's total CD4⁺ or CD8⁺T cell population (the population of lymphocytes in an individual isapproximately 3-5×10¹¹). The resulting T cell population can also begenetically transduced and used for immunotherapy or can be used inmethods of in vitro analyses of infectious agents. For example, apopulation of tumor-infiltrating lymphocytes can be obtained from anindividual afflicted with cancer and the T cells stimulated toproliferate to sufficient numbers. The resulting T cell population canbe genetically transduced to express tumor necrosis factor (TNF) orother proteins (for example, any number of cytokines, inhibitors ofapoptosis (e.g. Bcl-2), genes that protect cells from HIV infection suchas RevM10 or intrakines, and the like, targeting molecules, adhesionand/or homing molecules and any variety of antibodies or fragmentsthereof (e.g. Scfv)) and given to the individual.

[0163] In certain embodiments, the EMSP of the present invention can becontacted with paramagnetic particles such that said paramagneticparticles are engulfed by the EMSP. EMSP comprising paramagneticparticles can then be subjected to magnetic force and concentrated orlocalized to a particular site of interest, such as a tumor, site ofviral infection or site of autoimmune disease, and/or otherwiseselected, either in vitro or in vivo.

[0164] Likewise, the EMSP of the present invention can be used in vivoto stimulate tumor-specific T cells, autoantigen-sepcific T cells, andor viral-specific T cells. Within this context, in certain embodiments,the EMSP can be generated such that tolerance to such tumor, auto, orviral antigens is broken, either in an in vivo or in vitro setting. Inan in vivo setting, the EMSP can be administered locally to a tumorsite, a site of viral infection or site of autoimmune disease, oralternatively can be administered systemically.

[0165] One particular use for the CD4⁺ T cells populations of theinvention is the treatment of HIV infection in an individual. Prolongedinfection with HIV eventually results in a marked decline in the numberof CD4⁺ T lymphocytes. This decline, in turn, causes a profound state ofimmunodeficiency, rendering the patient susceptible to an array of lifethreatening opportunistic infections. Replenishing the number of CD4⁺ Tcells to normal levels may be expected to restore immune function to asignificant degree. Thus, the method described herein provides a meansfor selectively expanding CD4⁺ T cells to sufficient numbers toreconstitute this population in an HIV infected patient. It may also benecessary to avoid infecting the T cells during long-term stimulation orit may desirable to render the T cells permanently resistant to HIVinfection. There are a number of techniques by which T cells may berendered either resistant to HIV infection or incapable of producingvirus prior to restoring the T cells to the infected individual. Forexample, one or more anti-retroviral agents can be cultured with CD4⁺ Tcells prior to expansion to inhibit HIV replication or viral production(e.g., drugs that target reverse transcriptase and/or other componentsof the viral machinery, see e.g., Chow et al. Nature 361:650-653, 1993).

[0166] Several methods can be used to genetically transduce T cells toproduce molecules which inhibit HIV infection or replication. Forexample, in various embodiments, T cells can be genetically transducedto produce transdominant inhibitors, “molecular decoys”, antisensemolecules, intrakines, or toxins. Such methodologies are described infurther detail in U.S. patent application Ser. Nos. 08/253,751,08/253,964, and PCT Publication No. WO 95/33823, which are incorporatedherein by reference in their entirety.

[0167] The methods for stimulating and expanding a population of antigenspecific T cells are useful in therapeutic situations where it isdesirable to up-regulate an immune response (e.g., induce a response orenhance an existing response) upon administration of the T cells to asubject. For example, the method can be used either in vivo or in vitroto enhance a T cell response against tumor-associated antigens. Tumorcells from a subject typically express tumor-associated antigens but maybe unable to stimulate a co-stimulatory signal in T cells (e.g., becausethey lacks expression of co-stimulatory molecules). Thus, tumor cellscan be contacted with T cells from the subject in vitro and antigenspecific T cells expanded according to the method of the invention andthe T cells returned to the subject.

[0168] Accordingly, in one embodiment malignancies such as non-HodgkinsLymphoma (NHL) and B-cell chronic lymphocytic leukemia (B-CLL) can betreated. While initial studies using expanded T cells have been testedin NHL, (see Liebowitz et al., Curr. Opin. Onc. 10:533-541, 1998), the Tcell populations of the present invention offer unique phenotypiccharacteristics that can dramatically enhance the success ofimmunotherapy by providing increased engraftment (likely supplied bystimulation of the CD28 signal) and reactivity. However, patients withB-CLL present special difficulties, including low relative T cellnumbers with high leukemic cell burden in the peripheral blood,accompanied by a general T cell immunosuppression. The T cellpopulations of the present invention can provide dramatically improvedefficacy in treating this disease and especially when combined with stemcell transplantation therapy. Accordingly, increasing T cell functionand anti-CLL T cell activity with EMSP would be beneficial.

[0169] For example, given that deficient expression of CD154, the ligandfor CD40, on T cells of B-CLL patients has been cited as a majorimmunological defect of the disease, the T cell populations of thepresent invention, which may provide sustained high levels of CD154expression upon infusion, could aid in its treatment. Investigatorsreport that in CLL the capability of a patient's T cells' to expressCD154 is defective as well as the capability of the leukemic B-cells toexpress CD80 and CD86. The failure of leukemic B-cells in CLL toadequately express the ligands for CD28, could result in failure tofully activate tumor-responsive T cells and, therefore, may representthe mechanism underlying the T cells' apparent state of tolerance.Studies in which CD40 is engaged on CLL B cells, either via solubleanti-CD40 antibodies or via CD154-transduced leukemic B-cells, appearsto correct the defect in CD80 and CD86 expression and up-regulates MHCsurface expression. Kato et al., J. Clin. Invest. 101:1133-1141, 1998;Ranheim and Kipps, J. Exp. Med. 177:925-935, 1993. Cells treated in thisway were able to stimulate specific T cell anti-tumor responses.

[0170] With the enhanced expression of CD154 on the surface of the Tcell population of the present invention such T cells would be expectedto interact with autologous B-CLL cells, and would thus increase thattumor's immunogenicity by driving up expression of MHC, CD80, and CD86.This, in turn, should lead to a strong anti-tumor response. Further, oneof ordinary skill in the art would readily understand that treatment ofa patient with ex vivo expanded T cells of the present invention may becombined with traditional cancer therapies such as chemotherapy. In thisregard, for example, a patient may be treated with an agent such asFludarabine or Campath (Berlex Laboratories, Montville, N.J., USA),followed by infusion with T cell populations of the present invention orboth.

[0171] Alternatively, T cells can be stimulated and expanded asdescribed herein to induce or enhance responsiveness to pathogenicagents, such as viruses (e.g., human immunodeficiency virus), bacteria,parasites and fungi.

[0172] The invention further provides methods to selectively expand aspecific subpopulation of T cells from a mixed population of T cells. Inone embodiment, the invention provides specifically enriched populationsof T cells that have much higher ratio of CD4⁺ and CD8⁺ double positiveT cells. In an additional embodiment, the invention provides methods toselectively expand CD8+ T cells expressing increased levels of Bcl-xL.

[0173] Another embodiment of the invention, provides a method forselectively expanding a population of T_(H1) cells from a population ofCD4⁺ T cells. In this method, CD4⁺ T cells are co-stimulated with ananti-CD28 antibody, such as the monoclonal antibody 9.3, inducingsecretion of T_(H1)-specific cytokines, including IFN-γ, resulting inenrichment of T_(H1) cells over T_(H2) cells. In a further embodiment,methods are provided for selectively expanding Tc1 over Tc2 cells, orvice versa. Tc1 and Tc2 cells can be distinguished based on cytokinesecretion patterns using any number of assays known to the skilledartisan.

[0174] T cells have been demonstrated to be activated within a few hours(Iezzi et al., Immunity 8:89-95, 1998). Accordingly, in combination withthe methodologies herein described, this provides the ability to expanda tailor made subset of a T cell population in a short period of time.In one embodiment, this technique can be utilized at the bedside of asubject, in an outpatient modality, or at a subject's home, similar tothe use of kidney dialysis. For example, a method or device wherein Tcells are incubated in contact with activation signals (e.g., anti-CD3and anti-CD28 antibodies, and the like) and returned to the patientimmediately in a continuous flow or after a few hour expansion period.In one aspect, such techniques of expansion could use isolated chamberswith filter components, such that EMSP are mixed with a continuous flowof blood/concentrated cells. In another embodiment, EMSP within anapparatus may be provided to stimulate T cell activation and expansion.For example, a continuous fluid path from the patient through ablood/cell collection device and/or a disposable device containing EMSPand/or other components to stimulate T cells prior to cells returning tothe subject can be utilized. Such a system could involve a leukapheresisinstrument with a disposable set sterile docked to the existingmanufacturers disposable set, or be an adaptation to the manufacturer'sdisposable set. Further, the EMSP may be a part of a removal insertwhich is inserted into one of the device chambers or physically presentwithin one of the disposable components. In another embodiment of thecontinuous flow aspect discussed above, the system may comprisecontacting the cells with the activating components at room temperatureor at physiologic temperature using a chamber within a blood collectiondevice or an incubation chamber set up in series with the flow path tothe patient.

[0175] In another example, blood is drawn into a stand-alone disposabledevice directly from the patient that contains EMSP. In one embodiment,the disposable device may comprise a container (e.g., a plastic bag, orflask) with appropriate tubing connections suitable forcombining/docking with syringes and sterile docking devices. This devicewill contain a EMSP for immobilization of T cell activation components(e.g., anti-CD3 and anti-CD28 antibodies); Additionally when using thestand-alone device, the subject can remain connected to the device, orthe device can be separable from the patient. Further, the device may beutilized at room temperature or incubated at physiologic temperatureusing a portable incubator.

[0176] As devices and methods for collecting and processing blood andblood products are well known, one of skill in the art would readilyrecognize that given the teachings provided herein, that a variety ofdevices that fulfill the needs set forth above may be readily designedor existing devices modified. Accordingly, as such devices and methodsare not limited by the specific embodiments set forth herein, but wouldinclude any device or methodology capable of maintaining sterility andwhich maintains blood in a fluid form in which complement activation isreduced and wherein components necessary for T cell activation (e.g.,anti-CD3 and anti-CD28 antibodies or ligands thereto) may be separatedfrom the blood or blood product prior to administration to the subject.Further, as those of ordinary skill in the art can readily appreciate avariety of blood products can be utilized in conjunction with thedevices and methods described herein. For example the methods anddevices could be used to provide rapid activation of T cells fromcryopreserved whole blood, peripheral blood mononuclear cells, othercyropreserved blood-derived cells, or cryopreserved T cell lines uponthaw and prior to subject administration. In another example, themethods and devices can be used to boost the activity of a previously exvivo expanded T cell product or T cell line prior to administration tothe subject, thus providing a highly activated T cell product. Lastly,as will be readily appreciated the methods and devices above may beutilized for autologous or allogeneic cell therapy simultaneously withthe subject and donor.

[0177] The methods of the present invention may also be utilized withvaccines to enhance reactivity of the antigen and enhance in vivoeffect. Further, given that T cells expanded by the present inventionhave a relatively long half-life in the body, these cells could act asperfect vehicles for gene therapy, by carrying a desired nucleic acidsequence of interest and potentially homing to sites of cancer, disease,or infection. Accordingly, the cells expanded by the present inventionmay be delivered to a patient in combination with a vaccine, one or morecytokines, one or more therapeutic antibodies, or in combination withthe EMSP as described herein. Virtually any therapy that would benefitby a more robust T cell population is within the context of the methodsof use described herein.

[0178] Pharmaceutical Compositions

[0179] The EMSP and/or the target cell populations, such as T cellpopulations of the present invention may be administered either alone,or as a pharmaceutical composition in combination with diluents and/orwith other components such as IL-2 or other cytokines or cellpopulations. Briefly, pharmaceutical compositions of the presentinvention may comprise an EMSP or a target cell population as describedherein, in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions may comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.Compositions of the present invention are preferably formulated forintravenous administration.

[0180] Pharmaceutical compositions of the present invention may beadministered in a manner appropriate to the disease to be treated (orprevented). The quantity and frequency of administration will bedetermined by such factors as the condition of the patient, and the typeand severity of the patient's disease, although appropriate dosages maybe determined by clinical trials.

[0181] Pharmaceutical compositions of the present invention may beadministered in a manner appropriate to the disease to be treated (orprevented). The quantity and frequency of administration will bedetermined by such factors as the condition of the patient, and the typeand severity of the patient's disease, although appropriate dosages maybe determined by clinical trials.

[0182] The present invention also provides methods for preventing,inhibiting, or reducing the presence of a cancer or malignant cells inan animal, which comprise administering to an animal an anti-cancereffective amount of the subject EMSP with or without activated T cells.

[0183] The cancers contemplated by the present invention, against whichthe immune response is induced, or which is to be prevented, inhibited,or reduced in presence, may include but are not limited to melanoma,non-Hodgkin's lymphoma, Hodgkin's disease, leukemia, plasmocytoma,sarcoma, glioma, thymoma, breast cancer, prostate cancer, colo-rectalcancer, kidney cancer, renal cell carcinoma, pancreatic cancer,esophageal cancer, brain cancer, lung cancer, ovarian cancer, cervicalcancer, multiple myeloma, hepatocellular carcinoma, nasopharyngealcarcinoma, ALL, AML, CML, CLL, and other neoplasms known in the art.

[0184] Alternatively, compositions as described herein can be used toinduce or enhance responsiveness to pathogenic organisms, such asviruses, (e.g., single stranded RNA viruses, single stranded DNAviruses, double-stranded DNA viruses, HIV, hepatitis A, B, and C virus,HSV, CMV, EBV, HPV), parasites (e.g., protozoan and metazoan pathogenssuch as Plasmodia species, Leishmania species, Schistosoma species,Trypanosoma species), bacteria (e.g., Mycobacteria, Salmonella,Streptococci, E. coli, Staphylococci), fungi (e.g., Candida species,Aspergillus species) and Pneumocystis carinii.

[0185] The immune response induced in the animal by administering thesubject compositions of the present invention may include cellularimmune responses mediated by CD8+ T cells, capable of killing tumor andinfected cells, and CD4+ T cell responses. Humoral immune responses,mediated primarily by B cells that produce antibodies followingactivation by CD4+ T cells, may also be induced. A variety of techniquesmay be used for analyzing the type of immune responses induced by thecompositions of the present invention, which are well described in theart; e.g., Coligan et al., Current Protocols in Immunology, John Wiley &Sons Inc., 1994.

[0186] When “an immunologically effective amount,” “an anti-tumoreffective amount,” “a tumor-inhibiting effective amount,” or“therapeutic amount” is indicated, the precise amount of thecompositions of the present invention to be administered can bedetermined by a physician with consideration of individual differencesin age, weight, tumor size, extent of infection or metastasis, andcondition of the patient. It can generally be stated that apharmaceutical composition comprising the subject EMSP and/or activatedT cells, may be administered at a dosage to be determined duringappropriate clinical trials. EMSP compositions may also be administeredmultiple times at these dosages. The cells can be administered by usinginfusion techniques that are commonly known in immunotherapy (see, e.g.,Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimaldosage and treatment regime for a particular patient can readily bedetermined by one skilled in the art of medicine by monitoring thepatient for signs of disease and adjusting the treatment accordingly.

[0187] Activated T cells will be administered in dosages and routes andat times to be determined in appropriate clinical trials. T cellcompositions may be administered multiple times at dosages within theseranges. The EMSP-based method of therapy may be combined with othermethods, such as direct administration of the activated T cells of theinvention. The activated T cells and EMSP may be autologous orheterologous to the patient undergoing therapy. If desired, thetreatment may also include administration of mitogens (e.g., PHA) orlymphokines, cytokines, and/or chemokines (e.g., GM-CSF, IL-4, IL-13,Flt3-L, RANTES, MIP1-α, etc.) as described herein to enhance inductionof the immune response.

[0188] The administration of the subject pharmaceutical compositions maybe carried out in any convenient manner, including by aerosolinhalation, injection, ingestion, transfusion, implantation ortransplantation. The compositions of the present invention may beadministered to a patient subcutaneously, intradermally,intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.Preferably, the EMSP compositions of the present invention areadministered to a patient by intradermal or subcutaneous injection. TheT cell compositions of the present invention are preferably administeredby i.v. injection. The compositions of EMSP or activated T cells may beinjected directly into a tumor or lymph node.

[0189] In yet another embodiment, the pharmaceutical composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, Science 249:1527-33, 1990; Sefton, CRC Crit. Ref.Biomed. Eng. 14:201, 1987; Buchwald et al., Surgery 88:507, 1980; Saudeket al., N. Engl. J. Med. 321:574, 1989). In another embodiment,polymeric materials can be used (see Langer and Wise (eds.), MedicalApplications of Controlled Release, CRC Pres., Boca Raton, Fla., 1974;Smolen and Ball (eds.), Controlled Drug Bioavailability, “Drug ProductDesign and Performance,” Wiley, New York, 1984; Ranger and Peppas, J.Macromol. Sci. Rev. Macromol. Chem. 23:61, 1983; see also Levy et al.,,Science 228:190 1985; During et al., Ann. Neurol. 25:351, 1989; Howardet al., J. Neurosurg. 71:105, 1989). In yet another embodiment, acontrolled release system can be placed in proximity of the therapeutictarget, thus requiring only a fraction of the systemic dose (see, e.g.,Langer and Wise (eds.), Medical Applications of Controlled Release, CRCPres., Boca Raton, Fla., 1984, vol. 2, pp. 115-138).

[0190] The EMSP and T cell compositions of the present invention mayalso be administered using any number of matrices. Matrices have beenutilized for a number of years within the context of tissue engineering(see, e.g., Lanza, Langer, and Chick (eds.), Principles of TissueEngineering, 1997). The present invention utilizes such matrices withinthe novel context of acting as an artificial lymphoid organ to support,maintain, or modulate the immune system, typically through modulation ofT cells. Accordingly, the present invention can utilize those matrixcompositions and formulations which have demonstrated utility in tissueengineering. Accordingly, the type of matrix that may be used in thecompositions, devices and methods of the invention is virtuallylimitless and may include both biological and synthetic matrices. In oneparticular example, the compositions and devices set forth by U.S. Pat.Nos. 5,980,889; 5,913,998; 5,902,745; 5,843,069; 5,787,900; or 5,626,561are utilized, as such these patents are incorporated by reference intheir entirety. Matrices comprise features commonly associated withbeing biocompatible when administered to a mammalian host. Matrices maybe formed from both natural or synthetic materials. The matrices may benon-biodegradable in instances where it is desirable to leave permanentstructures or removable structures in the body of an animal, such as animplant; or biodegradable. The matrices may take the form of sponges,implants, tubes, telfa pads, fibers, hollow fibers, lyophilizedcomponents, gels, powders, porous compositions, or nanoparticles. Inaddition, matrices can be designed to allow for sustained release ofseeded cells or produced cytokine or other active agent. In certainembodiments, the matrix of the present invention is flexible andelastic, and may be described as a semisolid scaffold that is permeableto substances such as inorganic salts, aqueous fluids and dissolvedgaseous agents including oxygen.

[0191] A matrix is used herein as an example of a biocompatiblesubstance. However, the current invention is not limited to matrices andthus, wherever the term matrix or matrices appears these terms should beread to include devices and other substances which allow for cellularretention or cellular traversal, are biocompatible, and are capable ofallowing traversal of macromolecules either directly through thesubstance such that the substance itself is a semi-permeable membrane orused in conjunction with a particular semi-permeable substance.

[0192] In one aspect of the present invention, the EMSP described hereincan be used in vivo as an adjuvant as described in U.S. Pat. No.6,464,973. In a further embodiment, the EMSP can be used as a vaccine toinduce an immune response in vivo against an antigen of interest such asthose described herein (e.g. tumor antigens, viral antigens,autoantigens, etc). In one embodiment the EMSP can be used to generatean immune response in vivo, either administered alone or in combinationwith activated T cells as described herein or in combination with otherknown therapies.

[0193] In one embodiment of the present invention, EMSP may be used togenerate polyclonal and/or antigen-specific T cells in vitro or in vivo.T cells may be stimulated with EMSP loaded or engineered to expressantigen in the context of MHC as previously described. Such stimulationis performed under conditions and for a time sufficient to permit thegeneration of T cells that are specific for the antigen of interest. Forexample, T cells (5×10⁶ cells/ml) and antigen-loaded or expressing EMSP(2.5×10⁵ cells/ml) may be cultured in conventional media as describedherein, supplemented with 5-10% serum, 1 mM sodium pyruvate, with orwithour 100 IU/ml penicillin, with or without 100 μg/ml streptomycin,and 5×10⁻⁵ M β-mercaptoethanol in 96 well U-bottom plates at a ratio of20:1. After 5 days, cells may be tested for antigen-specificity in astandard 4 hours chromium release assay. Antigen-specific T cells may befurther expanded using techniques known in the art (as described in U.S.Pat. No. 5,827,642) or as described herein. Stimulation of T cells withEMSP as described in the Examples may be carried out following thestimulation with antigen-loaded EMSP to further increase expansion ofthe desired antigen-specific T cells.

[0194] All references referred to within the text are herebyincorporated by reference in their entirety. Moreover, all numericalranges utilized herein explicitly include all integer values within therange and selection of specific numerical values within the range iscontemplated depending on the particular use. Further, the followingexamples are offered by way of illustration, and not by way oflimitation.

EXAMPLES

[0195] Protocols

[0196] The constructions described below are carried out according tothe general techniques of genetic engineering and molecular cloningdetailed in, e.g., Maniatis et al., (Laboratory Manual, Cold SpringHarbor, Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The steps ofPCR amplification follow known protocols, as described in, e.g., PCRProtocols—A Guide to Methods and Applications (ed., Innis, Gelfand,Sninsky and White, Academic Press Inc. (1990)). Notably, theoligonucleotides used to modify the Ad genome may use differentrestriction enzyme sites than those identified below, and may use aslightly different insertion site in the genome, without affecting theoutcome of the invention. Such variations, so long as not substantial,are within the understanding of one of ordinary skill in the art.

[0197] Moreover, cells are transfected according to standard techniques,well known to a person skilled in the art. Protocols enabling a nucleicacid to be introduced into a cell may employ known methods, e.g.,calcium phosphate transfection (Maniatis et al., 1989), DEAE-dextrantechniques, electroporation, methods based on osmotic shocks,micro-injection of the selected cell, or methods based on the use ofliposomes.

[0198] Cloning and construction of cell-based artificial APC (aAPC).Human CD32 was cloned from neutrophils into the pcDNA 3.1 neo vector(Invitrogen, Carlsbad, Calif.), and transfected into K562 cells(American Type Culture Collection, Manassas, Va.) by electroporation;K32 cells were cloned by FACS sorting. Similarly, (h)4-1BB ligand wascloned from B cells into the pcDNA3.1 hygro vector (Invitrogen), andtransfected into K32 cells before FACS sorting.

[0199] CD8⁺ T lymphocyte preparation and K562 cell culture. Freshperipheral blood lymphocytes were obtained by leukopheresis andelutriation. CD4⁺ T cells were purified by negative selection using theOKT4 Ab (ATCC) as described by June el al., Mol. Cell Biol. 7:4472-4481(1987). (Ab=antibodies; mAb=monoclonal antibodies). CD8⁺ T cells werepurified identically, but OKT8 Ab (ATCC) was substituted for the OKT4Ab. All cultures were maintained in AIM V (GIBCO BRL, Life Technologies,Grand Island, N.Y.) with 3% human AB serum (BioWhittaker, Walkersville,Md.). Human IL-2 (Chiron Therapeutics, Emeryville, Calif.) was added at20 IU/mL where indicated.

[0200] T lymphocyte stimulation and long-term culture. At each timepoint at which the lymphocytes were stimulated, the K562 cell-basedaAPCs were irradiated with 10,000 rads, then washed twice into T cellculture medium. Cell-based aAPCs were then loaded with anti-CD3 (OKT3)and anti-CD28 mAbs (9.3) at 0.5 μg/ml for 10 minutes at roomtemperature. Unwashed, antibody-loaded aAPCs were then mixed with CD8⁺ Tcells at a 1:2 K562:T cell ratio. The T cell concentration wasmaintained at 0.5×10⁶ cells/ml throughout culture, and up to 100×10⁶ Tcells were maintained in flasks. Anti-CD3/28 bead stimulation wasperformed as previously described by Levine et al., J. Immunol.159:5921-5930 (1997). Cultured T cells were monitored for cell volumeand enumerated on a Coulter Multisizer II (Miami, Fla.) every 2-3 days,and re-stimulated at 7-10 day intervals when the mean lymphocyte volumereached 200-250 fL.

[0201] Flow cytometry and FACS sorting. Cells were stained withantibodies (and/or MHC tetramers) at 4° C., and analyzed on aFACSCalibur (BD BioSciences, Mountain View, Calif.). Apoptosis assayswere conducted per the manufacturer's protocol (R & D Systems,Minneapolis, Minn.). Cell sorting was performed on a MoFlo cell sorter(Cytomation, Fort Collins, Calif.). All flow cytometry data wereanalyzed with FlowJo software (TreeStar, San Carlos, Calif.).

[0202] Real-time PCR and TCR Vβ repertoire analysis. Real time PCR wasperformed and normalized to 28s rRNA levels as described previously byRiley et al., J. Immunol. 166, 4943-4948 (2001). The diversity of TCR Vβrepertoire was assessed by determination of CDR3 size lengths bymultiplex PCR as previously described by Claret et al., J. Clin. Invest.100:855-866 (1997).

[0203]⁵¹Cr release assays. Target T2 cells (ATCC) were pulsed with 10 μMflu peptide (see Maus et al., 2002) or left unpulsed before labelingwith ⁵¹chromium (PerkinElmer Life Sciences, Inc., Boston Mass.). After afour-hour incubation of effectors with targets, radioactivity wascounted from an aliquot of supernatant. Specific lysis was calculated bystandard methods.

[0204] After a four-hour incubation of effectors with targets,radioactivity was counted from an aliquot of supernatant. Specific lysiswas calculated by standard methods.

Example 1 Construction of Artificial APCs (aAPCs)

[0205] A cell-based aAPC was designed which could be geneticallymanipulated to express different co-stimulatory molecules in addition toCD28. K562 cells were chosen because they do not express HLA proteinsthat would promote allogeneic responses, but they do express the T cellinteraction molecules ICAM (CD54) and LFA-3 (CD58) (FIG. 1A). K562 cellsexpressing the human Fcγ receptor CD32 (K32 cells) were transfected andthen cloned to permit exogenous loading of anti-CD3 and anti-CD28antibodies (FIG. 1A). Similarly, the K32/4-1BBL line (FIGS. 1A, B) wasgenerated by transfecting K32 cells with human 4-1BB ligand. Cultureswere initiated by adding γ-irradiated aAPCs to fresh human CD8⁺ T cellsprepared by negative selection as described.

[0206] K32 and K32/4-1BBL aAPCs efficiently activate human polyclonalCD8⁺ T cells. The aAPCs were tested for their ability to stimulate theinitial activation and proliferation of primary CD8⁺ T cells. The Tcells were stimulated with three different preparations of aAPCs: CD3/28beads, K32 cells coated with anti-CD3 and anti-CD28 (K32/CD3/28), orK32/4-1BBL cells coated with the same antibodies (K32/4-1BBL/CD3/28).The initial rate of growth of the T cells stimulated with all threeaAPCs was equivalent, as judged by thymidine incorporation (FIG. 1C).This observation was confirmed by labeling fresh T cells withcarboxyfluorescein diacetate succinimidyl ester (CFSE) and tracking celldivision during the first five days of culture (data not shown). TheK562 cell-based system was found to be equivalent to CD3/28 beads forthe induction of proliferation and cell division of CD4⁺ T cells (FIG.1C and data not shown). Neither K562-based aAPCs, nor CD8⁺ T cells, norCD4⁺ T cells incubated separately showed any proliferation (FIG. 1C anddata not shown). Thus, the requirements for the initial rounds of CD8⁺ Tcell proliferation were satisfied equally by CD3/CD28 stimulationprovided in the context of polystyrene beads or cell based aAPCs, andthe addition of 4-1BBL co-stimulation did not appear to have furtherbenefit.

Example 2 K32/4-1BBL aAPCs Permit Long-Term Expansion of HumanPolyclonal CD8⁺ T Cells

[0207] Next, to determine whether the aAPC were sufficient to maintainlong term propagation of CD8⁺ T cells (FIG. 2A) CD8 T cells werestimulated with aAPCs—but no exogenous cytokines were added to themedium. CD3/28 bead-stimulated cells failed to proliferate after thesecond stimulation with aAPCs, in agreement with previous findings.Similarly, CD8⁺ T cells stimulated with CD3/28 in the context of K32cells entered into a plateau phase of the growth curve within 2 weeks ofculture, and no additional net growth of cells occurred afterre-stimulation.

[0208] In contrast, when CD8⁺ T cell cultures were stimulated withK32/4-1BBL/CD3/28 aAPCs, they remained in exponential growth even aftera third stimulation. This augmentation of long-term proliferation wasreproducible, as the average increase in the total number of T cells was410-fold higher in cultures stimulated with K32/4-1BBL/CD3/28 than incultures stimulated with CD3/28 beads in six independent experiments.

[0209] Phenotypic analysis of cultures showed a progressive enrichmentfor CD3⁺CD8⁺ T cells after stimulation with K32/4-1BBL/CD3/28 aAPCs(FIG. 2B). The cell based aAPCs rapidly disappeared from the cellculture, as evidenced by an inability to detect the irradiatedK32/4-1BBL cells by flow cytometry after seven days (FIG. 2C). Thisfinding was confirmed in large-scale experiments and also by RT-PCR forCD32 (data not shown). Thus, the mixed T cell and aAPC culture yields apopulation of essentially pure T cells within one week.

Example 3 Efficient Propagation of Antigen-Specific Cytotoxic T Cells byK32/4-1BBL aAPCs

[0210] Because in certain embodiments, immunotherapy with CD8⁺ T cellswill likely require cells with antigen-specific cytolytic functions, itwas necessary to determine whether the K32/4-1BBL aAPCs could be used toexpand antigen-specific CTLs, although antigens are not essential in thepresentation of the aAPCs. Consequently, they were used to culture apopulation of MHC tetramer sorted primary CD8⁺ T cells for 10 weeks(FIG. 3A). Purified CD8⁺ T cells obtained from an HLA-A*0201 donor werestained and sorted with an A*0201 MHC tetramer loaded with a flu matrixprotein peptide (flu MP tetramer). The tetramer⁺ population was presentat an initial frequency of 0.081% (FIG. 3B), which presumably wascomposed mainly of memory T cells. Cultures of tetramer⁻ CD8⁺ T cellsserved as an internal control population of T cells to assess the growthpotential and specificity of the tetramer⁺ population of CD8⁺ T cells.

[0211] After bulk sorting, 16,000 cells each of CD8⁺fluMP-tetramer⁺ andtetramer⁻ phenotype were stimulated with irradiated K32/4-1BBL/CD3/28aAPCs (FIG. 3C). All cells were re-stimulated with K32/4-1BBL aAPCs at10 day intervals and rhIL-2 (20 IU/mL) was added to the culture duringthe ⁴ ^(th) week. No specific flu stimulation Was provided duringculture. Exponential growth curves of both populations of cells wereobtained for several months. The 16,000 antigen-specific T cells yielded1.5×10⁹ cells after one month of culture, a number of cells sufficientfor immunotherapy. The substantial proliferative capacity of the CD8⁺ Tcells that remained after 30 days of culture indicated that these CTLscould have substantial long-term engraftment potential after adoptivetransfer.

[0212] To determine if antigen specificity, of the expanded populationswas maintained during culture, cells were stained with flu MP tetramer(FIG. 3B). On day 17, the population that was initially sorted as flu MPtetramer⁺ was 61.7% CD8⁺flu MP tetramer⁺, while the population that wassorted as flu MP tetramer had negligible staining. The percentage oftetramer⁺ cells in culture declined somewhat over time, but remainedat >20% through day 60 (data not shown).

[0213] Similar results were obtained with T cells from another HLAA*0201 donor, where on day 26 of culture, the population sorted as fluMP tetramer⁺ was 49% CD8⁺ flu MP tetramer⁺ and again remained at >20%through day 60 (data not shown). Thus, a single round of selection forCD8⁺ cells with the desired specificity is sufficient to maintainacceptable purity of CD8⁺ cells cultured on K32/4-1BBL/CD3/28 aAPCs.

[0214] To examine the effector function of the cultured T cells, theantigen-specific cytolytic activity of the flu MP tetramer⁺ andtetramer⁻ cultures was determined by ⁵¹Cr release assays on days 26, 30,and 56 of ex vivo expansion (FIG. 3D and data not shown). The HLA-A*0201TAP deficient T2 cell line, pulsed or unpulsed with the flu MP peptidewas used as a target population. At all time points, flu MP tetramer⁺cells displayed potent cytotoxicity for flu-MP peptide pulsed targets.Flu MP tetramer⁺ cells did not kill unpulsed targets, and the Flu MPtetramer⁻ cells did not kill either pulsed or unpulsed target cells.Neither effector population killed the parental K562 cells, suggestingthat killing was MHC-restricted, and not directed at K562 alloantigens(data not shown). Similar results were obtained with both donors (datanot shown).

Example 4 Maintenance of Diverse TCR Repertoire by K32/4-1BBL aAPC

[0215] Given the finding that many tumor antigens are self antigens,adoptive immunotherapy will require the isolation and propagation of Tcells with generally low affinity TCRs. Therefore, it is desirable thatthe culture system propagate T cells with uniform efficiency. To comparethe properties of the cultures grown with aAPCs, cultures of enrichedCD8⁺ T cells grown on anti-CD3/28 coated beads, and K32/CD3/28 andK32/4-1BBL/CD3/28 aAPCs were assessed for maintenance of the initial TCRrepertoire. CDR3 size length analysis of TCR β-chains was used becauseit permits sensitive detection of clonal T cell outgrowth.

[0216] It has been previously shown by the inventors that CD3/28 coatedbeads can maintain diverse CD4+ T cell populations for several months inculture. However, dramatic perturbations of the input CD8 repertoireoccurred after two weeks of culture on these beads. In contrast,enriched CD8⁺ T cells cultured on K32/4-1BBL/CD3/28 aAPC maintained CDR3size length distributions that were similar to the input population of Tcells (FIG. 4). The addition of 4-1BBL appeared to account for thepreservation of the repertoire, because cultures of enriched CD8⁺ Tcells on K32/CD3/28 aAPC did not maintain a comparably diverserepertoire (FIG. 4).

Example 5 K32/4-1 BBL aAPC Stimulation Enhances Survival of Human CD8⁺ TCells upon Re-Stimulation

[0217] Because the initial growth rate of CD8⁺ T cells stimulated withthree different aAPCs was similar, it appeared that the increasedoverall growth observed in K32/4-1BBL/CD3/28 stimulated T cells was dueto improved survival. Therefore, a determination was made of therelative effects of the various aAPCs on Bcl-xL and IL-2 expression, twogenes involved in T cell survival and proliferation, respectively.

[0218] Quantitative real time RT-PCR was used to determine the levels ofsteady-state mRNA coding for Bcl-xL and IL-2 (FIG. 5). In all cultures,Bcl-xL and IL-2 gene expression was upregulated compared to restingcells one and three days after the first stimulation, and by day 10,Bcl-xL and IL-2 gene expression had returned to resting levels. However,one to three days after re-stimulation, only CD8⁺ T cell cultures thatwere stimulated with the K32/4-1BBL/CD3/28 aAPCs had increased levels ofBcl-xL and IL-2 mRNA. In contrast, CD8⁺ T cells that were stimulatedwith beads or K32/CD3/28 cells did not re-induce Bcl-xL or IL-2expression after a second stimulation (FIGS. 5A and B, respectively).Together these data suggest that 4-1BB co-stimulation provides asurvival signal that is critical for subsequent but not the initialstimulation of CD8⁺ T cell proliferation.

[0219] The viability was assessed of the CD8⁺ T cells stimulated by thevarious aAPCs during culture by fluorescent staining with annexin V andpropidium iodide (FIG. 6). In the bead-stimulated cultures, viabilitygradually decreased in the first ten days, and then droppedprecipitously as only 6% of cells were viable on day 20. In the T cellcultures stimulated with K32 aAPCs, T cell viability seven days afterthe second stimulation was improved compared to bead-stimulated cells.However, most of the cells died by day 20.

[0220] In contrast, K32/4-1BBL/CD3/28 stimulated CD8⁺ T cell cultureswere >70% viable throughout culture. Together these results show thatthe addition of 4-1BBL co-stimulation prevents apoptosis and preservesthe starting repertoire of CD8⁺ T cells.

[0221] In sum, the K562 cell based aAPC system is able to maintain longterm exponential growth of viable T cells, particularly CD8⁺ memorycells for many months in vitro. Based on a starting cell population of10⁴ influenza specific CD8⁺ T cells, a sufficient number of CTL wereobtained for therapy after only 30 days of culture. Since the startingnumber of antigen specific CD8⁺ T cells could be isolated from only 100ml of blood, given an initial frequency of 0.05%, it would be possibleto decrease the culture time to only two weeks by performing aleukapheresis and isolating 10⁵ to 10⁶ antigen-specific CD8⁺ T cells.High speed cell sorting or magnetic bead separation can isolatesufficient CD8⁺ memory cells for initial culture on K32/4-1BBL aAPCcoated with anti-CD3 and CD28 antibodies. Alternatively, it is possibleto coat the K32/4-1BBL aAPC with the desired tetramer in order toculture antigen specific T cells de novo, and obviate the need for aseparate cell isolation procedure. The flexibility of the present systemis particularly advantageous, in that the engineering of the aAPC can bemodified and focussed based upon the specific T cell need.

[0222] One implication of the present system is that the CTLs retain asubstantial replicative capacity after culture with theK32/4-1BBL/CD3/28 aAPCs, even after reaching therapeutic numbers forclinical infusion. Several mechanisms appear to account for the improvedgrowth and repertoire of K32/4-1BBL/CD3/28 stimulated CD8⁺ T cells. Forinstance, as noted, there was a markedly improved survival of CD8⁺ Tcells after repeated stimulation with K32/4-1BBL/CD3/28 aAPC, ascompared with CD3/28 coated beads. With the addition of 4-1BBco-stimulation, CD8⁺ T cells have increased expression of IL-2 andBcl-xL, improved survival, and continued proliferation afterre-stimulation with anti-CD3/CD28. Thus, 4-1BB stimulation in thiscontext overcomes the previously described activation-inducednon-responsiveness.

[0223] Not all clinically useful antigens are presently characterized asMHC-restricted epitopes, and the library of MHC tetramers for many HLAtypes remains limiting. Therefore, K32/4-1BBL/CD3/28 aAPCs were alsoused to expand CTLs that have been previously enriched for a particularantigen-specificity by priming with autologous DC that have been pulsedwith apoptotic bodies of autologous tumor (unpublished data). Thus,K32/4-1BBL/CD3/28 aAPCs are likely to be complementary to many methods,including MHC tetramer sorting (Dunbar et al., Curr. Biol. 8:413-416(1998); Yee, et al., J. Immunol. 162:2227-2234 (1999)), or priming withautologous DCs or other artificial APCs (Latouche et al., 2000), thatenrich for antigen-specific CTL populations. Although thus far theK32/4-1 BBL/CD3/28 aAPCs have been tested for their ability to expandmemory or primed T cells; they and other APC constructs will be usefulto expand naive CD8⁺ cells as a source of the ‘self’ repertoire fortumor immunotherapy (Curtsinger et al., J. Immunol. 160:3236-3243(1998); Sagerstrom et al., Proc. Natl. Acad. Sci. USA 90:8987-8991(1993); Wang et al., J. Immunol. 164:1216-1222 (2000).

[0224] Expanding low-avidity, self-reactive T cells Voltz et al., N.Engl. J. Med. 340:1788-1795 (1999) that can differentiate into memorycells (Tan, J. Clin. Invest. 108:1411-1415 (2001)) offers a usefulapproach to derive therapeutic numbers of self reactive CTLs.Advantageously, because only T cells that recognize the MHC/peptidecomplex are activated in the present invention, rapid expansion isprovided for selected antigen specific clones. Once characterized, thesecell lines will be invaluable tools for immunotherapy, particularlysince the cell lines permit the design of optimal co-stimulation regimeson a disease-by-disease basis. Moreover, given that GMP preparations ofanti-CD3 and CD28 antibodies are currently available, and thatK32/4-1BBL aAPC can be grown in serum free medium, the system of thepresent invention provides therapeutic resources for clinical adoptiveimmunotherapy for patients with cancer and viral diseases, as well asfor the in vitro propagation of CTLs for experimentation. Finally, inlight of the many co-stimulatory molecules that continue to bediscovered, e.g., OX40L, CD40, CD80, CD86, GL50, 4-1BBL and B7-H1, thatserve to either augment the level of T cell growth or alter thefunctional ability of the T cells, the present invention offers novelmethods by which the usefulness of these additional co-stimulators canbe evaluated as immunotherapeutic agents by transfecting them into K-32cells and testing their effect on overall T cell growth and infunctional assays.

Example 6 K32 aAPCs With and Without CD86 Permit Long-Term Expansion ofHuman Polyclonal CD4⁺ T Cells

[0225] Construction and initial testing of K32 aAPCs with and withoutCD86. We wanted to design an artificial APC (aAPC) that would allow therapid expansion of human CD4 T cells in an antigen and MHC independentmanner. To evaluate different human cell lines in their ability tostimulate CD4 T cell growth, we transfected K562 and U937 cells withCD32 with and without CD86 to create K32, U32 and K32/86 cell lines.Both of these myelogenous leukemia cell lines grow in suspension and thetransition of K562 cells, in particular, to clinical trials will beexpedited because they do not express MHC molecules that would promotean allogenic response, can easily be killed by natural killer (NK)cells, and grow well in serum free conditions.

[0226] To initially characterize the ability of these cell lines tostimulate T cell proliferation, we performed a standard [³H]-thymidineincorporation assay. Irradiated K32+anti-CD3 and CD28 Abs (K32/CD3/28),U32+ anti-CD3 and CD28 Abs (U32/CD3/28), K32/86+anti-CD3 Ab cells(K32/86/CD3) and anti-CD3 and CD28 Abs coated beads (CD3/28 coatedbeads) were used to stimulate freshly isolated human CD4 T cells and[³H]-thymidine incorporation was measured after three days of culture.All cell based aAPC stimulated cultures demonstrated higher[³H]-thymidine uptake than the cells stimulated with CD3/28 coated beadsdemonstrating that at the level of inducing T cell proliferation, cellbased expansion systems were more potent than the bead based system.Control cultures in which the anti-CD3 and anti-CD28 Ab were left outdemonstrated minimal (background) levels of [³H]-thymidine, indicatingthat CD4 T cells rather than the irradiated stimulator cells wereresponsible for the [³H]-thymidine uptake. K32/CD3/28 and CD3/28 coatedbead stimulated cells continued to grow exponentially for ten dayswithout restimulation. Additionally, CD4 T cells stimulated withK32/CD3/28 or K32/CD3/86 underwent on average two more populationdoublings within the first ten days indicating that it is a more rapid Tcell expansion system than the CD3/28 coated beads.

[0227] For optimal engraftment potential and possible therapeuticbenefit, it is important to ensure that the T cells, after in vitroexpansion, are functional and not senescent at the time of re-infusion.To test whether CD4 T cells expanded by K32/CD3/28 aAPCs were able toproduce cytokines and survival factors upon restimulation, fresh CD4 Tcells were stimulated with either K32/CD3/28 or CD3/28 coated beads andallowed to expand for 10 days. Three days after restimulation, RNA washarvested and cytokine production was measured by quantitative RT-PCR.We observed that CD4 T cells restimulated with K32 CD3/28 could induce awide array of cytokines (IL-2, IL-10, and IFNγ), a costimulatorymolecule (ICOS) and a cell survival factor (Bclx-L) in all cases greaterthan or equal to as cells stimulated with CD3/28 coated beads.

[0228] Furthermore, log linear growth of CD4 T cells was maintained forat least 45 days using the K32/CD3/28, K32/86/CD3 and CD3/28 coated beadexpansion systems with all of the cultures undergoing at least two morerestimulations demonstrating K32 CD3/28 stimulated cells have thecapacity to expand far beyond what is required for immunotherapy trials.At the end of 45 days of culture the K32/CD3/28 stimulated CD4 T cellshad undergone 26 population doublings (6.7×10⁷ fold expansion, data notshown). These studies demonstrate that rapid expansion of CD4 T cellscan be achieved using K32/CD3/28 aAPCs and suggest that once these cellsare infused back into the patient, they will be at least as functionalas CD4 T cells stimulated by CD3/28 coated beads.

[0229] One possible use of ex vivo expanded polyclonal T cells is toreconstitute the immune system of immunodeficient individuals. For thistherapy to be successful, gaps in the T cell repertoire must not becreated by selective expansion of certain T cell subtypes. As describedabove in Example 4, using Vβ T cell repertoire analysis it was foundthat CD8 T cells expanded with K32/4-BBL/CD3/28 were not skewed to anyparticular Vβ family. These findings were extended using a backcalculation method described by Wells et al (Wells,A. D.,Gudmundsdottir,H., and Turka,L. A., Following the fate of individual Tcells throughout activation and clonal expansion. Signals from T cellreceptor and CD28 differentially regulate the induction and duration ofa proliferative response. J. Clin.Invest 100, 3173-3183, 1997) thatmeasures the number of cells under each peak to determine the percentageof cells that never divided. Table 1 below shows the calculationsperformed to elucidate what percentage of the resting CD4 T cellsstimulated with either K32/CD3/28 or CD3/28 coated beads that divided.Our results from this analysis indicate that upon optimal stimulationwith an aAPC the vast majority (95% for K32/CD3/28 and 90% for CD3/28coated bead stimulated cells) of all human CD4 T cells can divide. TABLE1 K32/CD3/28 CD3/28 Coated Beads % of Starting % of Starting TotalAbsolute Population in Absolute Population in Division Number Number ofEach Total Number Number of Each Division Peak of Cells PrecursorsDivision Peak of Cells Precursors Peak 0 106 106 5 213 213 10 1 555 27814 916 458 22 2 2506 626 31 2322 580 28 3 5144 643 32 3694 462 22 4 5466342 17 5534 346 16 5 558 17 1 1519 47 2

[0230] Table Legend: The vast majority of human CD4 T cells divide uponoptimal stimulation. CD4 T cells were labeled with CFSE and stimulatedwith either K32/CD3/28 or CD3/28 coated beads as described above. Afterfour days of stimulation, the number of cells under each division peakwas determined using Flow-Jo software. The absolute number of precursorswas determined by dividing the total number of cells by 2^(n) wheren=the number of divisions (division peak). The percentage of startingpopulation in each division peak was determined by dividing the absolutenumber of precursors by the total number of absolute precursors (2012,for K32/CD3/28 and 2016 for CD3/28 coated beads) (Wells,A. D.,Gudmundsdottir,H., and Turka,L. A., Following the fate of individual Tcells throughout activation and clonal expansion. Signals from T cellreceptor and CD28 differentially regulate the induction and duration ofa proliferative response. J.Clin.Invest 100, 3173-3183, 1997).

Example 7 Co-Culture with CD3/28 Activated CD4 T Cells InducesUpregulation of mRNA Encoding for IL-15, B7-H1, B7-D and B7-H3 in K32aAPC

[0231] Co-culture with CD3/28 activated CD4 T cells induces upregulationof mRNA encoding for IL-15, B7-H1, B7-D and B7-H3 in K32. Molecules thatwere preferentially expressed in K32 cells but not U32 cells that mayaccount for their differences to serve as aAPCs and to augment IL-2production. We hypothesized that crosstalk between K32/CD3/28 aAPCs andthe recently activated CD4 T cells were inducing the expression ofcostimulatory molecules on the irradiated K32 cells. Therefore, weassayed for the expression of molecules in the aAPC/CD4 mixtures in thepresence or absence of anti-CD3 and anti-CD28 antibodies, allowing us tocompare expression of costimulatory molecules and cytokines in thepresence or absence of activated CD4 T cells. Li et al. demonstratedthat IL-15 is critical for the onset of T cell division in a murinemodel (Li,X. C., Demirci,G., Ferrari-Lacraz,S., Groves,C., Coyle,A.,Malek,T. R., and Strom,T. B., IL-15 and IL-2: a matter of life and deathfor T cells in vivo. Nat.Med. 7, 114-118, 2001.), making it a candidateresponsible for the early onset of cell division in K32/CD3/28 aAPCsstimulated CD4 T cells. K32 cells constitutively express low levels ofmRNA encoding for IL-15. After coculture with activated CD4 T cells,IL-15 mRNA was upregulated 15-fold. U32 cells did not express IL-15 mRNAeither constitutively or after incubation with activated CD4 T cells. Inaddition to cytokines, differential expression of costimulatory cellsurface molecules could also account for the differences in stimulatorycapacities between K32 and U32 aAPCs. We did not detect expression ofOx40L or B7-H1 (ICOS ligand) by RT-PCR in either K32 or U32. Using flowcytometry, we were unable to detect expression of CD80 or 41B B-L oneither K32 or U32 and could not detect CD86 on K32 cells. U32 cellsexpress low levels of CD86. K562 cells do express high levels of ICAM-1and LFA-3 (Maus,M. V., Thomas,A. K., Leonard,D. G., Allman,D., Addya,K.,Schlienger,K., Riley,J. L., and June,C. H., Ex vivo expansion ofpolyclonal and antigen-specific cytotoxic T lymphocytes by artificialAPCs expressing ligands for the T cell receptor, CD28 and 4-1BB.Nat.Biotechnol. 20, 143-148, 2002), but blocking experiments withmonoclonal antibodies specific for both ICAM-1 and LFA-3 failed todiminish CD4 cell proliferation, demonstrating that these adhesionmolecules are not required for the strong T cell stimulation by K32cells.

[0232] Next, we searched for expression of newly described costimulatorymolecules PD-L1, PD-L2 and B7-H3, whose role in human T cell activationhas not been clearly established. While we did not detect constitutiveexpression PD-L1 in K32, U32, or resting CD4 T cells, we did observea >45 fold upregulation of PD-L1 mRNA K32/CD3/28 stimulated CD4 Tcultures that was not observed in U32/CD3/28 stimulated culture.Likewise, we found a low level of PD-L2 mRNA in resting K32 cells thatwas modestly upregulated upon K32/CD3 stimulation. Much higherquantities of PD-L2 mRNA were observed in K32/CD3/28 stimulatedcultures. Minimal PD-L2 expression was detected in CD4 T cells mixedwith U32/CD3 or U32/CD3/28 aAPCs.

[0233] Lastly, we searched for expression of another recently describedcostimulatory molecule, B7-H3 (Chapoval,A. I., Ni,J., Lau,J. S.,Wilcox,R. A., Flies,D. B., Liu,D., Dong,H., Sica,G. L., Zhu,G.,Tamada,K., and Chen,L., B7-H3: a costimulatory molecule for T cellactivation and IFN-gamma production. Nat.Immunol. 2, 269-274, 2001.).Unlike PD-L1 and PD-L2, B7-H3 was constitutively expressed severalthousand-fold over resting CD4 T cells in K32 cells as compared to U32aAPCs. Coculture with anti-CD3 and/or anti-CD28 did not significantlychange B7-H3 expression in K562 cells suggesting it is unlikely to be areason K32/CD3/28 stimulated CD4 T cells expand longer than K32/CD3activated CD4 T cells; however, it could explain why K32/CD3 aAPC (i.e.anti-CD28 deficient aAPC) can induce CD4 T cells to produce IL-2 andexpand. To demonstrate that, in fact, the K32 cells were expressingIL-15, PD-L1, PD-L2 and B7-H3 rather than the CD4 T cells, we treatedirradiated K32 with supernatant from T cells activated with K32/CD3/28or CD3/28 coated beads. We observed similar levels of induction of IL-15and PD-L1 in K32 cells stimulated with supernatants from K32/CD3/28 orCD3/28 bead activated T cells demonstrating that cell contact betweenthe K562 cell and the T cell is not necessary for the upregulation ofthese molecules and soluble factors can substitute for activated Tcells. B7-H3 was only modestly upregulated in K32 cells after incubationwith supernatant from K32/CD3/28 stimulated T cells consistent withminimal upregulation observed. PD-L2 was only slightly induced on K32cells after incubation with T cell supernatant suggesting that eithercell-to-cell contact is necessary to upregulate PD-L2 or a substantialfaction of the PD-L2 mRNA upregulation occurred in the T cells. Theunexpected expression of these recently described costimulatory ligandsin K32 cells may contribute their potency as aAPC.

[0234] All of the above U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, including butnot limited to U.S. patent application Ser. No. 09/960,264, filed Sep.20, 2001; which is a continuation-in-part of U.S. application Ser. No.09/794,230, filed Feb. 26, 2001; which claims the benefit of ProvisionalApplication Nos. 60/184,788, filed Feb. 24, 2000, and 60/249,902, filedNov. 17, 2000, are incorporated herein by reference, in their entirety.

[0235] From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims. All of references, patents,patent applications, etc. cited above, are incorporated herein in theirentirety. Further, all numerical ranges recited herein explicitlyinclude all integer values within the range.

What is claimed is:
 1. An engineered multivalent signaling platform(EMSP) for use in stimulating and/or activating T cells comprising anEMSP that expresses or displays on its surface one or more agents thatligate a cell surface moiety of at least a portion of T-cells andstimulates said T-cells.
 2. The EMSP of claim 1 wherein said platformcomprises a human cell line.
 3. The EMSP of claim 2 wherein said humancell line is selected from the group consisting of K562, U937, 721.221,T2, and C1R cells.
 4. The EMSP of claim 3 wherein said human cell lineis K562.
 5. The EMSP of claim 1 wherein said EMSP is a cell that isgenetically modified to express a human Fcγ receptor.
 6. The EMSP ofclaim 5 wherein said human Fcγ receptor comprises CD32.
 7. The EMSP ofclaim 1 wherein said EMSP is a cell that is genetically modified toexpress CD32 and said one or more agent is an antibody that binds to acell surface molecule on the surface of T cells.
 8. The EMSP of claim 7wherein said EMSP is further genetically modified to express aco-stimulatory molecule.
 9. The EMSP of claim 8 wherein saidco-stimulatory molecule is selected from the group consisting of CD80,CD86, 4-1BBL, OX40L, ICOS-L, ICAM, PD-L1 and PD-L2.
 10. The EMSP ofclaim 7 wherein said one or more agent is an antibody that is displayedon the surface of said EMSP via interaction with the Fcγ receptor. 11.The EMSP of claim 7 wherein said one or more agent is a natural ligandfor a cell surface molecule on the surface of a T cell.
 12. The EMSP ofclaim 11 wherein said natural ligand comprises the natural ligand forCD28.
 13. The EMSP of claim 11 wherein said natural ligand comprises4-1BBL.
 14. The EMSP of claim 1 wherein said EMSP is a cell that hasbeen modified to express a cytokine.
 15. The EMSP of claim 14 whereinsaid cytokine is selected from the group consisting of IL-2, GM-CSF,IL-4, TNF-α, and IFN-γ.
 16. The EMSP of claim 1 wherein said T cellscomprise CD4+ T cells.
 17. The EMSP of claim 1 wherein said T cellscomprise CD8+ T cells.
 18. The EMSP of claim 1 wherein said T cellscomprise regulatory T cells.
 19. The EMSP of claim 10 wherein saidantibody is an anti-CD28 antibody.
 20. The EMSP of claim 10 wherein saidantibody is an anti-CD3 antibody.
 21. The EMSP of claim 10 wherein saidantibody comprises anti-CD3 and anti-CD28.
 22. The EMSP of claim 1wherein said one or more agent comprises at least an antigen.
 23. TheEMSP of claim 22 wherein said antigen is selected from the groupconsisting of a tumor antigen, a bacterial antigen, a fungal antigen, aviral antigen, and an autoantigen.
 24. A method for activating orstimulating a population of T-cells by cell surface moiety ligation,comprising: a. providing a population of cells wherein at least aportion thereof comprises T-cells; b. contacting said population ofcells with an EMSP, said EMSP having on its surface one or more agentsthat ligate a cell surface moiety of at least a portion of said T-cellsand activates or stimulates said T-cells.
 25. The method of claim 24,wherein said EMSP comprises a cell.
 26. The method of claim 25, whereinsaid cell is a human cell.
 27. The method of claim 26, wherein said cellis a human cell line.
 28. The method of claim 24, further comprisingexpanding said T-cells by incubating in the presence of EMSP.
 29. Themethod of claim 24, further comprising separating said T-cells from saidEMSP and subsequently incubating said T-cells with an agent thatfacilitates T-cell expansion.
 30. The method of any one of claims 24-28,wherein the T-cell is a T-cell line.
 31. The method of any one of claims24-28, wherein said T-cell is a T-cell clone.
 32. A method formaintaining T-cell repertoire comprising providing a population of cellshaving T-cells present therein, exposing said T-cells to an EMSP for atime sufficient to induce activation and subsequently expanding saidT-cells.
 33. The method of claim 32, wherein said expansion occurs inthe absence of said EMSP.
 34. The method of claim 32, wherein saidexpansion occurs in the presence of said EMSP.
 35. A method formaintaining the viability of T-cells during culture, comprisingcontacting said T-cells with EMSP.
 36. The method of claim 35, whereinsaid T-cells are CD4+ T-cells.
 37. The method of claim 35, wherein saidT-cells are CD8+ T-cells.
 38. The method of claim 37, wherein saidcontacting occurs by sequential stimulation of T-cells by an EMSP. 39.The method of claim 38, wherein the viability of said T-cells is greaterthan 50% after at least one, two, three, four, five, six, seven, oreight rounds of stimulation.
 40. The method of claim 38, wherein theviability of said T-cells is greater than 60% after at least one, two,three, four, five, six, seven, or eight rounds of stimulation.
 41. Themethod of claim 38, wherein the viability of said T-cells is greaterthan 70% after at least one, two, three, four, five, six, seven, oreight rounds of stimulation.
 42. The method of claim 38, wherein theviability of said T-cells is greater than 80% after at least one, two,three, four, five, six, seven, or eight rounds of stimulation.
 43. Amethod for decreasing apoptosis of a population of CD8+ T-cells,comprising stimulating said cells with EMSP, said EMSP having on itssurface at least one primary stimulatory agent and at least oneco-stimulatory agent.
 44. The method of claim 43, wherein said CD8+T-cells are expanded in the presence of said EMSP.
 45. The method ofclaim 43, wherein said CD8+ T-cells are expanded in the absence of saidEMSP.
 46. The method of claim 43, wherein said CD8+ T-cells demonstratean increase in Bcl-XL levels compared to CD8+ T-cells expanded in theabsence of initial stimulation with said EMSP.
 47. The method of any oneof claims 43-46, wherein at least one co-stimulatory agent comprises4-1BB ligand.
 48. The method of claim 47, wherein the primarystimulatory agent comprises an agent that binds CD3.
 49. The method ofclaim 48, wherein said EMSP further comprises a second co-stimulatoryagent that binds CD28.
 50. The method of any one of claims 43-49,further comprising at least one sequential round of restimulation.
 51. Amethod for expanding a population of T-cells by cell surface moietyligation, comprising: a. providing a population of cells wherein atleast a portion thereof comprises T-cells; b. contacting said populationof cells with an EMSP, said EMSP having on its surface one or moreagents that ligate a cell surface moiety of at least a portion of saidT-cells and activates or stimulates said T-cells; and c. culturing saidT-cells under conditions and time sufficient to induce cell division,wherein said contacting and/or said culturing occurs in the absence ofexogenously added cytokines.
 52. The method of claim 51, wherein saidEMSP comprises a cell.
 53. The method of claim 51, wherein said cell isa human cell.
 54. The method of claim 53, wherein said cell is a humancell line.
 55. The method of claim 51, further comprising sequentiallystimulating said T-cells with EMSP either by previously purifyingT-cells from originally added EMSP and subsequently adding additionalEMSP or by adding additional EMSP to previously stimulated cells withoutseparation of originally added EMSP.
 56. The method of claim 51, furthercomprising separating said T-cells from said EMSP and subsequentlyincubating said T-cells with an agent that facilitates T-cell expansion,followed by restimulation with EMSP.
 57. The method of any one of claims51-56, wherein the T-cell is a T-cell line.
 58. The method of any one ofclaims 51-56, wherein said T-cell is a T-cell clone.
 59. The method ofany one of claims 51-58, wherein said EMSP comprises a cell displaying aligand for any one of CD3, CD28, or 4-1BB.
 60. A method for expanding apopulation of T-cells by cell surface moiety ligation, comprising: a.providing a population of cells wherein at least a portion thereofcomprises T-cells; b. contacting said population of cells with an EMSP,said EMSP having on its surface one or more agents that ligate a cellsurface moiety of at least a portion of said T-cells and activates orstimulates said T-cells; and c. culturing said T-cells under conditionsand time sufficient to induce cell division.
 61. The method of claim 60,wherein said EMSP comprises a cell.
 62. The method of claim 60, whereinsaid cell is a human cell.
 63. The method of claim 62, wherein said cellis a human cell line.
 64. The method of claim 60, further comprisingsequentially stimulating said T-cells with EMSP either by previouslypurifying T-cells from originally added EMSP and subsequently addingadditional EMSP or by adding additional EMSP to previously stimulatedcells without separation of originally added EMSP.
 65. The method ofclaim 60, further comprising separating said T-cells from said EMSP andsubsequently incubating said T-cells with an agent that facilitatesT-cell expansion, followed by restimulation with EMSP.
 66. The method ofany one of claims 60-65, wherein the T-cell is a T-cell line.
 67. Themethod of any one of claims 60-65, wherein said T-cell is a T-cellclone.
 68. The method of any one of claims 60-67, wherein said EMSPcomprises a cell displaying a ligand for any one of CD3, CD28, or 4-1BB.69. The method of claim 60, wherein following initial stimulation oractivation said T-cells are separated from EMSP and then expanded in thepresence of exogenously added cytokines.
 70. The method of claim 69,wherein said T-cells are substantially free of EMSP prior to expansion.71. A population of T-cells expanded by the method of claim 51 or 60,wherein said T-cells are substantially free of EMSP.
 72. A method ofimmunotherapy comprising administering an EMSP to a subject in needthereof, thereby stimulating a population of T-cells within the subjectto expand.
 73. A method of enhancing reactivity to an antigen comprisingadministering an EMSP to a subject in need thereof concurrently, priorto or following inoculation of the subject with an antigen of interest,thereby stimulating a population of antigen specific T-cells within thesubject to expand.
 74. A method for breaking immune tolerance comprisingadministering an EMSP to a subject either systemically or locally at asite of interest.
 75. A method for increasing uptake of an exogenouslyadded nucleic acid molecule in T-cells, comprising contacting saidT-cells with an EMSP and contacting said T-cells with said nucleic acidmolecule thereby, said contacting of EMSP with said T-cells renderingcells more amenable to uptake of nucleic acid.
 76. The method of claim75, wherein said exogenously added nucleic acid is operably linked to apromoter
 77. The method of claim 76, wherein said nucleic acid moleculeprovides gene replacement for abnormal gene product.
 78. The method ofany one of claims 24-70 wherein the natural functionality of saidT-cells is preserved following stimulation and expansion
 79. A method ofactivating antigen specific T-cells, comprising contacting a populationof T-cells an antigen and an EMSP under conditions and for timesufficient to induce activation of T-cells specific to said antigen. 80.The method of claim 79, wherein said activation occurs ex vivo afterwhich the T-cells are administered to a patient substantially free or incombination with said EMSP.
 81. The method of claim 79, wherein saidantigen is displayed by said EMSP.
 82. The method of claim 81, whereinsaid display is by way of a fusion protein.
 83. The method of claim 79,wherein said antigen is in the form of a complex with MHC derivedpeptides.
 84. The method of claim 29, wherein said agent thatfacilitates T-cell expansion comprises IL-2.